La marchitez bacteriana de la papa (MB) causada por Ralstonia solanacearum (Smith, 1896) (Yabuuchi et al., 1995), es una de las más devastadoras enfermedades que afectan el cultivo de la papa en los países en desarrollo. En la actualidad, no existe un control químico efectivo. De todas las medidas de control, la utilización de variedades genéticamente resistentes es la mejor estrategia de manejo de la enfermedad y la que ofrece mejores perspectivas. El objetivo principal de este estudio fue identificar genotipos de especies silvestres de papa que presenten altos niveles de resistencia a la MB con énfasis en la resistencia a la infección latente en tubérculos. Para ello, se evaluaron 4461 genotipos diferentes de papa silvestre del Banco de Germoplasma del Centro Internacional de la Papa (CIP). 10 plantas por genotipo fueron inoculadas con una suspensión de la cepa CIP204 de R. solanacearum a una concentración de 1 x 108 ufc/g de suelo. Para asegurar la durabilidad de la resistencia, los genotipos resistentes fueron evaluados nuevamente contra 7 cepas de R. solanacearum con alta agresividad. Los genotipos resistentes a por lo menos 5 cepas fueron sometidos a una tercera prueba donde se evaluó además la resistencia a la infección en tubérculos en condiciones menos severas. Se encontraron 178 genotipos resistentes a la cepa CIP204 (4% de los tamizados). De 162 genotipos resistentes analizados, la resistencia pudo ser confirmada en sólo 52 genotipos en tamizados consecutivos con varias cepas del patógeno, de los cuales 9 genotipos también son resistentes al tizón tardío de la papa. De 26 genotipos analizados para la infección en tubérculos, 7 genotipos (6 de Solanum acaule y 1 de Solanum chacoense) fueron resistentes a la marchitez en planta y no mostraron infección latente ni en tallos ni en tubérculos. S. acaule y S. chacoense son las especies más promisorias para los futuros programas de mejoramiento. Esta es la primera evidencia de que altos niveles de resistencia a la marchitez bacteriana existe en la naturaleza. Palabras clave: MB, papa, resistencia, silvestres, infección latente. / The Bacterial Wilt of the Potato(BW) caused by Ralstonia solanacearum (Smith, 1896) (Yabuuchi et al., 1995), is one of the most devastating illnesses that affect the potato’s cultivation in the developing countries. At the present time an effective chemical control does not exist. Of all the control measures of BW, the use of genetically resistant varieties is the best handling strategy of the illness and the one that offer better perspectives. The main objective of this study was to identify genotypes of wild potato species that show high levels of resistance to bacterial wilt with emphasis in the resistance to the latent infection in tubers. For that reason, 4461 different genotypes from the Germoplasm Bank at the International Potato Center (CIP) Lima-Perú,were evaluated. Ten plants per genotype were inoculated with a suspension of the strain CIP204 of R. solanacearum to a concentration of 108 ufc/g soil. To ensure the durability of resistance, resistant genotypes were tested against seven strains of R. solanacearum with various levels of aggressiveness. The genotypes resistant to at least 5 strains of R. solanacearum were re-exposed to the pathogen in less severe conditions to assess the presence of latent infection in tubers. 178 genotypes (4%) of all screened were found to be resistant to CIP204 strain. From162 resistant genotypes analyzed, resistance could be confirmed in only 52 in subsequent screenings with several strains of the pathogen. From these, 9 genotypes are also resistant lo Late Blight. From 28 genotypes tested for tuber infection, seven (6 of S. acaule and 1 of S. chacoense) were found resistant to plant wilt and did not harbour any stem or tuber latent infection. S. acaule and S. chacoense are the most promising species for future breeding programs.This is the first evidence that high resistance levels of resistance to Bacterial Wilt exist in the nature. Keywords: BW, potato, resistance, wild, latent infection

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Alimentos. / Made available in DSpace on 2013-07-15T22:41:58Z (GMT). No. of bitstreams: 1 211457.pdf: 782449 bytes, checksum: d435cd88c2395a1093a9452faf9e9c9a (MD5) / Poli(3-hidroxibutirato), P(3HB), é um polímero de reserva de carbono e energia acumulado intracelularmente por diversos microrganismos, sob condições desbalanceadas de crescimento. Além da vantagem de ser biodegradável e biocompatível, possui propriedades termoplásticas compatíveis aos plásticos convencionais. Embora apresente estas vantagens, apresenta um elevado custo de produção frente aos polímeros petroquímicos, o que dificulta o seu uso. Neste trabalho estudou-se a produção de P(3HB) em resíduos de indústrias de alimentos, objetivando reduzir os custos de produção. Primeiramente, verificou-se a capacidade de crescimento da bactéria Ralstonia eutropha nos resíduos das indústrias amiláceas e indústrias processadoras de maçã. Testou-se três modelos primários de crescimento, e o modelo Logístico, foi o que melhor descreveu o crescimento da bactéria nas diferentes fontes de carbono testadas. Após verificar a capacidade de crescimento da bactéria, foi realizada a produção de P(3HB), nos resíduos das indústrias de alimentos, e também a suplementação do meio de cultura com ácido oléico e óleo de soja, visando aumentar a produção de polímero. O resíduo da indústria amilácea mostrou-se uma boa fonte de carbono para a produção de P(3HB), onde os conteúdos de polímero acumulado foram de 46, 50 e 56% com produtividades de 0,22; 0,37 e 0,49 g.L-1.h-1 para as culturas sem suplementação, suplementação com ácido oléico e suplementação com óleo de soja. Quando se realizou o crescimento no resíduo da indústria processadora de maçã os conteúdos de P(3HB) acumulados foram de 14, 34 e 22% do total da massa celular seca, para as culturas sem suplementação, suplementação com ácido oléico e suplementação com óleo de soja. Apesar do baixo conteúdo de polímero acumulado, a utilização do bagaço de maçã como fonte de carbono para a produção de polihidroxialcanoatos é uma boa alternativa, pois é uma matéria-prima barata que pode contribuir na redução dos custos de produção deste biopolímero. A adição dos suplementos nutricionais ao meio de cultura favoreceu a produção de P(3HB) aumentando o conteúdo de polímero acumulado e a produtividade.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-10-03T14:14:43Z No. of bitstreams: 1 Adonis Queiroz Mendes.pdf: 1093724 bytes, checksum: bc45e45f52c149dd3de62f8015c94732 (MD5) / Made available in DSpace on 2017-10-03T14:14:43Z (GMT). No. of bitstreams: 1 Adonis Queiroz Mendes.pdf: 1093724 bytes, checksum: bc45e45f52c149dd3de62f8015c94732 (MD5) Previous issue date: 2017-07-28 / Among the Solanaceae family, tomato plant is the second most produced in the world, behind only the potato. In Northeast of Brazil, its cultivation has been limited to sub regions of the Agreste and the Sertão, as a result of plant diseases problems, especially bacterial wilt. It is a disease caused by a complex of Ralstonia solanacearum species, and it has been reported in more than 450 species of plants. The losses caused by this disease are severe in plantations occurred during rainy summers and inside of greenhouses. This study aims to identify and obtain tomato germplasms resistant to bacterial wilt from 23 lines of tomato and 24 experimental hybrids. It was utilized the isolates CRM 74, CRM 76 and CRM 77 of Ralstonia pseudosolanacearum for the evaluation of lines, and the isolates CRM 74 and CRM 77 for evaluation of the hybrids. The 23 lines, with seven controls, were sowed, transplanted after 21 days and inoculated with 15 ml of the bacterial suspension with 5x108 CFU ml-1 per vase of 500 ml. The experiment had a completely randomized with three repetitions and every parcel had four vases with one plant each. Evaluations were carried out observing the incidence and severity of the disease and based on the result it was calculated: bacterial wilt index (IMB), incidence (INC), latency period (PL 50) and area below the curve of disease progression (AACPD). The data were submitted to analysis of variance and the means were grouped by the Scott-Knott test at 5% probability. Besides that estimating the phenotypic, genotypic and environmental correlation coefficients between the resistance components and calculating the dissimilarity of the Mahalanobis distance (D) to determine the genetic divergence among the lines. The Yoshimatsu and Hawaii 7996 were used as testers in crosses with the 10 lines of best results tomatoes and cultivars IPA-6 and Santa Clara. Subsequently, F1 hybrid was evaluated for bacterial wilt resistance during 15 days, analyzing the incidence and disease severity (SEV) using a descriptive scale notes from 0 to 4. The data were used to determine the general (CGC) and specific combining (CEC) capacities to four resistance components, estimating genetic parameters such as heritability, phenotypic, genotypic and environmental variance. The interaction genotypes x isolates presented significant difference at 1% probability only for the variables latency period and area below the disease progress curve. Considering bacterial wilt index, lines L04, L42, L49, L53, L82, L120, L125 and L128 were classified as resistant, but for incidence there were no lines that behaved similarly to the resistant controls Hawaii 7996, Yoshimatsu and Woodstock. As for genotype correlations, in 100% of the pairs of characters, values equal to or slightly higher than the phenotypic correlations were observed. Similar to these two, in 50% of the cases, were slightly higher than the environmental correlations, showing that the environment favored as the same form for IMB x PL 50, IMB x AACPD and PL 50 x AACPD. For the analysis of the dendrogram, a cut around 15.2% dissimilarity allowed the formation of four distinct groups and four subgroups. Group I was composed of resistant genotypes containing: Hawaii 7996, Woodstock, Yoshimatsu, Tropithai, L04, L42, L49, L53, L82, L125, L120 and L128, in addition to lines with moderate resistance. Considering the combining abilities of the experimental hybrids, the crosses that showed the greatest potentials for resistance were the YOS x L04 with the two isolates, and the HAW x L125 with the isolate CRM 74, since they were the only ones with a positive CEC for PL 50 and negative for the others characters. Also, presenting values from intermediate to superior that highlighted for the character PL 50, besides a high value for CEC, and at least one of the parents presented a high CGC value, which it is desirable. Observing the genetic parameters, the estimated values of the genetic variance were higher than the environmental variance for all the resistance components studied for the two bacterial isolates. However, it was observed in this study that the gene expression of the tomato resistance phenotype is resulted from the action of additive and non-additive gene effects, where the non-additive gene effects are involved in the four resistance components and that the additive effects are involved in the IMB, INC and AACPD. / Dentre as solanáceas o tomateiro é a segunda mais produzida no mundo, perdendo apenas para a batata. No Nordeste brasileiro o seu cultivo tem se limitado às mesorregiões do Agreste e do Sertão, tendo como um dos fatores para os problemas fitossanitários, principalmente a murcha bacteriana. Trata-se de uma doença causada pelo complexo de espécies Ralstonia solanacearum, sendo já relatada em mais de 450 espécies de plantas. As perdas causadas por essa doença são severas em plantios realizados durante verões chuvosos e em cultivo protegido. Este estudo teve como objetivo identificar e obter germoplasmas de tomateiro resistentes à murcha bacteriana entre 23 linhagens e 24 híbridos experimentais de tomateiro. Foram utilizados os isolados CRM 74, CRM 76 e CRM 77 de Ralstonia pseudosolanacearum para avaliação das linhagens e os isolados CRM 74 e CRM 77 para avaliação dos híbridos experimentais. As 23 linhagens, juntamente com sete testemunhas, foram semeadas, transplantadas após 21 dias e inoculadas com 15 ml de suspensão bacteriana na concentração de 5x108 UFC ml-1 para cada vaso de 500 ml. O experimento foi realizado em delineamento inteiramente casualizado com três repetições e cada parcela foi constituída por quatro vasos com uma planta cada. As avaliações foram realizadas observando a incidência e a severidade da doença e a partir dessas medidas foram calculados: índice de murcha bacteriana (IMB), incidência (INC), período de latência (PL 50) e área abaixo da curva de progresso da doença (AACPD). Os dados foram submetidos à análise de variância e as médias agrupadas pelos testes de Scott-Knott, a 5% de probabilidade, além de estimar os coeficientes de correlações fenotípicas, genotípicas e ambientais entre os componentes de resistência e calcular a dissimilaridade através da distância de Mahalanobis (D2) determinando a divergência genética entres as linhagens. As cultivares Yoshimatsu e Hawaii 7996 foram utilizadas como testadoras em cruzamentos com as cultivares IPA-6 e Santa Clara, além de 10 linhagens de tomateiro que apresentaram os melhores resultados na etapa anterior. Posteriormente, os híbridos F1 foram avaliados quanto à resistência à murcha bacteriana durante 15 dias, quanto à incidência e a severidade da doença (SEV) com auxílio de escala descritiva de notas variando de 0 a 4. Os dados foram utilizados para determinar a capacidade geral (CGC) e específica (CEC) de combinação para os quatro componentes de resistência, além de estimar parâmetros genéticos, tais como herdabilidade, variância fenotípica, genotípica e ambiental. A interação genótipos x isolados apresentou diferença significativa a 1% de probabilidade apenas para as variáveis período de latência e área abaixo da curva de progresso da doença. Considerando o índice de murcha bacteriana, as linhagens L04, L42, L49, L53, L82, L120, L125 e L128 foram classificadas como resistentes, mas para incidência não houve linhagem que se comportasse de forma semelhante às testemunhas resistentes Hawaii 7996, Yoshimatsu e Woodstock. No tocante às correlações genotípicas, em 100% dos pares de caracteres, foram observados valores iguais ou ligeiramente superiores às correlações fenotípicas, assim como essas duas, em 50% dos casos, foram ligeiramente superiores às correlações ambientais, mostrando que o ambiente favoreceu da mesma forma para IMB x PL 50, IMB x AACPD e PL 50 x AACPD. Para análise do dendrograma, um corte em torno de 15,2% de dissimilaridade possibilitou a formação de quatro grupos distintos e quatro subgrupos. O grupo I foi constituído por genótipos considerados resistentes: Hawaii 7996, Woodstock, Yoshimatsu, Tropithai, L04, L42, L49, L53, L82, L120, L125 e L128, além de linhagens com resistência moderada. Considerando as capacidades de combinação dos híbridos experimentais, os cruzamentos que apresentaram os maiores potenciais para resistência foram os YOS x L04 com os dois isolados e o HAW x L125 com o isolado CRM 74, pois foram os únicos com CEC positiva para PL 50 e negativa para os demais caracteres, apresentando valores de intermediário a superior e que se destacaram para a variável PL 50, além de alto valor para CEC, pelo menos um dos genitores apresentou alto valor de CGC, o que é desejável. Observando os parâmetros genéticos, os valores estimados da variância genética foram superiores aos da variância ambiental para todos os componentes de resistência estudados para os dois isolados bacterianos. Todavia, observou-se neste estudo que a expressão gênica do fenótipo resistência em tomateiro é resultante da ação de efeitos gênicos aditivos e não aditivos, em que os efeitos gênicos não aditivos estão envolvidos nos quatro componentes de resistência e que os efeitos aditivos estão envolvidos no IMB, INC e AACPD.

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