Please read the abstract in the section 00front of this document / Dissertation (MSc)--University of Pretoria, 2008. / Plant Science / unrestricted

Studies performed on natural pathosystems revealed that the spatial structure of the metapopulation plays an important role in the development and spread of disease; phytopathological experimental studies have also revealed a significant effect of climatic factors on the presence of the disease (incidence) and the level of infection (prevalence) in populations. The aim of this study was to explore a natural pathosystem from both sides at the same time and to see which local and spatial environmental factors influence the development of pathosystem. Study focuses on population biology of natural pathosystem Podospaera balsaminae - Impatiens noli-tangere, which has not been investigated yet. In the study area (between the city Sázava (Benešov district) and village Vlkančice) host populations occurred in 78 (64 of them were infected) in 2013 and 82 populations (61 of them were infected) in 2014. Size of host populations had the strongest effect to incidence and prevalence of disease in the pathosystem. In larger populations, the disease occurred more often, developed faster and was higher. The rate of spread of the pathogen territory and the rate of population host connectivity had also strong effect. The abundance of pathogen in the studied area and the level of connectivity of host populations also...

Les maladies infectieuses sont souvent étudiées à l'aide d'approches réductionnistes alors qu'elles sont fortement influencées par de nombreux facteurs hôtes et environnementaux en interaction. Ainsi, de nombreuses maladies d’étiologie complexe restent difficiles à caractériser. En développant une approche holistique pour aborder la complexité de l'interaction, (i) nous avons déchiffré les interactions complexes sous-jacentes au syndrome de mortalité des huîtres du Pacifique chez les juvéniles d’huîtres Crassostrea gigas, la principale espèce d'huître exploitée dans le monde et (ii) nous avons validé le mécanisme de pathogénèse quel que soit l’environnement infectieux et le génotype de l’huître. En utilisant une expérience d’infection écologiquement réaliste combinée à des analyses moléculaires (métabarcoding, transcriptomique, et suivi des agents pathogènes) et histologiques sur des familles d'huîtres aux susceptibilités contrastées à la maladie, nous avons démontré que la maladie est causée par une infection multiple avec comme première étape nécessaire l’infection des cellules immunocompétentes de l’huître (les hémocytes) par OsHV-1µvar. La réplication du virus induit un état immunodéprimé de l’huître qui conduit à une septicémie par des bactéries pathogènes opportunistes entraînant la mort des huîtres. En identifiant les interactions intra-hôtes entre les microorganismes et l'immunité de l'hôte, cette étude déchiffre le code du syndrome de mortalité des huîtres du Pacifique et fournit d'importantes données pour la conception de mesures prophylactiques et de programmes de sélection d'huîtres résistantes au syndrome de mortalité. Nous pensons qu'une telle approche de la biologie des systèmes pourrait être appliquée pour déchiffrer d'autres maladies multifactorielles qui affectent des espèces d'invertébrés non modèles dans le monde entier. / Infectious diseases are very often explored using reductionist approaches, despite repeated evidence showing them to be strongly influenced by numerous interacting host and environmental factors. Many diseases with complex etiology therefore remain misunderstood. In this thesis, by developing a holistic approach to tackle the complexity of the interaction, (i) we deciphered the complex intra-host interactions underlying the Pacific oyster mortality syndrome affecting juveniles of Crassostrea gigas, the main oyster species exploited worldwide and (ii) we validated this mechanism in different infectious environments and oyster genotypes. Using ecologically realistic experimental infections combined with thorough molecular (metabarcoding, transcriptomics, pathogen monitoring) and histological analyses on oyster families with contrasting susceptibilities, we demonstrated that the disease is caused by a multiple infection whose initial and necessary step is the infection of oyster haemocytes by a herpesvirus. Viral replication leads to an immune-compromised state of the host, evolving toward subsequent bacteremia by opportunistic bacteria. By identifying critical intra-host interactions between microorganisms and host immunity, this study cracks the code of the Pacific oyster mortality syndrome and provides important molecular data for the design of prophylactic measures and breeding programs dedicated to the production of oysters resistant to the mortality syndrome. We believe that such a systems biology approach could be applied to decipher other multi-factorial diseases that affect non-model invertebrate species worldwide.

Submitted by Tatiany Aparecida Teixeira Soratto (tatiany_soratto@hotmail.com) on 2017-10-31T12:37:37Z No. of bitstreams: 2 DISSERTACAO_Tatiany_final.pdf: 3171924 bytes, checksum: 81e554e8e0137d88785acf8a80455d32 (MD5) Encaminhamento do orientador.pdf: 236358 bytes, checksum: 344f7f279720c6058970c50ae22f4a46 (MD5) / Approved for entry into archive by Alini Demarchi (alini@cca.ufscar.br) on 2017-11-30T14:04:32Z (GMT) No. of bitstreams: 2 DISSERTACAO_Tatiany_final.pdf: 3171924 bytes, checksum: 81e554e8e0137d88785acf8a80455d32 (MD5) Encaminhamento do orientador.pdf: 236358 bytes, checksum: 344f7f279720c6058970c50ae22f4a46 (MD5) / Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-01-15T17:25:13Z (GMT) No. of bitstreams: 2 DISSERTACAO_Tatiany_final.pdf: 3171924 bytes, checksum: 81e554e8e0137d88785acf8a80455d32 (MD5) Encaminhamento do orientador.pdf: 236358 bytes, checksum: 344f7f279720c6058970c50ae22f4a46 (MD5) / Made available in DSpace on 2018-02-01T13:19:19Z (GMT). No. of bitstreams: 2 DISSERTACAO_Tatiany_final.pdf: 3171924 bytes, checksum: 81e554e8e0137d88785acf8a80455d32 (MD5) Encaminhamento do orientador.pdf: 236358 bytes, checksum: 344f7f279720c6058970c50ae22f4a46 (MD5) Previous issue date: 2017-09-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The Centro APTA Citros Sylvio Moreira/IAC has been conducting an extensive breeding program of citrus via directed crosses. In a previous study with Citrushuanglongbing pathosystem (HLB) held in our group, using a population obtained by hybridization between Citrus sunki and Poncirus trifoliata, differences were found in the multiplication of the bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB, in the parents and in the progeny. It was observed that the rate of infection and bacterial concentration was higher in C. sunki than in P. trifoliata. Thus, it is important to deepen the studies with this genus and hybrids to increase knowledge of which mechanisms could be involved in the tolerance to HLB, considered the most important disease of citrus currently. In this sense, the objective of this study was to establish sinteny between the linkage groups of the C. sunki and P. trifoliata maps with the genome of Citrus sinensis and to map genomic regions associated to tolerance CLas bacterium through phenotypic analysis (QTLs) and gene expression (eQTLs). With the comparative analysis between maps and genome, it was observed that all the linkage groups showed synteny with reference genome chromosomes used, with the exception of the linkage group 10 of the C. sunki map. For the phenotypic data, a population of 79 F1 hybrids between C. sunki and P. trifoliata was used. The quantification of the bacterium and accumulation of starch in the leaves were evaluated after two years of inoculation with the pathogen. Through the statistical analysis of the mixed model it was possible to group the hybrids into resistant, tolerant and susceptible, being important the validation of these data in the field. The expression of 14 candidate genes related to HLB was performed in 72 hybrids of the population and used as expression data for the mapping of eQTLs. It was possible to locate nine QTLs and 52 eQTLs on the C. sunki genitor map and 17 QTLs and 40 eQTLs were found on the P. trifoliata genitor map. The overlapping eQTLs of the majority genes of QTL (phenotypic data) indicates that the genes are related to the phenotype and are probably responsive to the pathogen infection. / O Centro APTA Citros Sylvio Moreira/IAC vem realizando um amplo programa de melhoramento genético de citros via cruzamentos dirigidos. Em um estudo prévio com o patossistema Citros-huanglongbing (HLB) realizado pelo nosso grupo, utilizando uma população obtida por hibridação controlada entre Citrus sunki e Poncirus trifoliata, foram verificadas diferenças na multiplicação da bactéria Candidatus Liberibacter asiaticus (CLas), agente causal do HLB, tanto nos genitores quanto na progênie. A taxa de infecção e a concentração de bactéria foi maior em Citrus sunki em relação ao P. trifoliata. Assim, é importante aprofundar os estudos com esses gêneros e seus híbridos para ampliar o conhecimento de quais mecanismos poderiam estar envolvidos na tolerância ao HLB, considerada a mais importante doença dos citros atualmente. O objetivo do trabalho foi estabelecer sintenia entre os grupos de ligação dos mapas de C. sunki e P. trifoliata com o genoma de Citrus sinensis e mapear regiões genômicas associadas à tolerância a CLas por meio de análise fenotípica (QTLs) e de expressão gênica (eQTLs). Com a análise comparativa entre mapas e genoma, foi observado que todos os grupos de ligação apresentaram sintenia com pseudocromossomos do genoma de referência utilizado, com exceção do grupo de ligação 10 do mapa da C. sunki. Para os dados fenotípicos foi utilizada uma população de 79 híbridos F1 entre C. sunki e P. trifoliata, sendo avaliada a quantificação da bactéria e acúmulo de amido nas folhas após dois anos da inoculação com o patógeno. Com a análise estatística utilizando modelo misto foi possível agrupar os híbridos em resistentes, tolerantes e suscetíveis, sendo importante a validação desses dados em campo. A análise de expressão de 14 genes candidatos relacionados ao HLB foi realizada em 72 híbridos da população e utilizados como dados de expressão para o mapeamento de eQTLs. Foram encontrados nove QTLs e 52 eQTLs no mapa do genitor C. sunki enquanto no mapa do genitor P. trifoliata foram encontrados 17 QTLs e 40 eQTLs. A sobreposição de eQTLs da grande maioria dos genes com QTLs dos dados fenotípicos, indicam que os genes têm relação com o fenótipo e que provavelmente são responsivos à infecção do patógeno.

Variable mortality of Pyrenophora semeniperda--infected Bromus tectorum seeds has been referred to as a "race for survival", stating that seeds that germinate quickly are more likely to escape pathogen-caused mortality. Dormancy status is not the only variable determining outcomes within the Bromus-Pyrenophora pathosystem. Varying temperature and exposure to water may strongly influence germination outcomes of B. tectorum when in the presence of P. semeniperda. Low water potentials characteristic of semi-arid soils are often over-looked in the context of seed pathogens, and are ecologically relevant- especially for plant species that inhabit intermittently dry environments. To adequately characterize the Bromus tectorum-Pyrenophora semeniperda pathosystem, four studies were conducted to address the following questions: (1) do temperature, water potential, and dormancy status influence germination outcomes in the Bromus-Pyrenophora pathosystem, (2) do repeated wetting-drying scenarios influence germination outcomes of infected B. tectorum seeds following dehydration at low water potentials similar to those found in the field (i.e., -4 through -150 MPa), (3) can we accurately characterize the asexual life cycle of P. semeniperda on a dormant B. tectorum seed, determining when infection takes place, and what occurs during disease development in continuously hydrated conditions, and (4) how does disease development of P. semeniperda influence the B. tectorum seed embryo and endosperm. All studies were conducted using dormant and/or non-dormant B. tectorum seeds and an intermediate strain of P. semeniperda. Study one used varying temperatures (5-20°C), and five water potentials (0, -0.5, -1, -1.5, -2 MPa) (achieved using PEG 8000). Inoculated seeds were exposed to low water potentials at various temperatures for 7, 14, 21, or 28 days then re-hydrated for 28 days. In the second study, seeds were incubated at 20°C at four nominal water potentials (-4, -10, -40, or -150 MPa) following 8 or 24 hours of initial hydration. Seeds were dehydrated for 1, 7, 14, or 21 days, then re-hydrated. In study three, inoculated seeds were chemically fixed between days 0 and 21 and viewed with a scanning electron microscope. In the fourth study, infected seeds were frozen with liquid nitrogen following 3, 8, and 14 days of disease development, then cross sectioned longitudinally and laterally prior to chemical fixation. Results indicate that non-dormant seeds escape death by germinating rapidly under favorable conditions, that incubation at low water potentials greatly increases seed mortality, that -10 MPa is near the threshold for full pathogen activity, and at water potentials lower than -40 MPa, P. semeniperda may successfully survive severe dehydration if previous hydration resulting in infection has occurred. SEM images indicate that mycelia penetration occurs within 8-24 hours, and that mycelium may penetrate all opening in the seed (i.e., stomata, cracks). Development of P. semeniperda is shown to cause significant damage to the endosperm and embryo within 8 days. As starch is consumed, the endosperm collapses leaving a hollow middle. The embryo is more resilient, but gradually deforms and deteriorates.

Variable mortality of Pyrenophora semeniperda–infected Bromus tectorum seeds has been referred to as a “race for survival.” Dormant seeds are highly susceptible to P. semeniperda infection. While much is known about primary dormancy little is known about secondary dormancy in B. tectorum seeds. Dormancy status is not the only variable determining outcomes within the Bromus - Pyrenophora pathosystem. Varying temperature and intermittent hydration may strongly influence germination outcomes of B. tectorum in the presence of P. semeniperda. While it has long been assumed that B. tectorum seeds are infected by P. semeniperda in the fall it was recently suggested that seeds may be infected in the summer; however, there is little evidence to support this. To further characterize the Pyrenophora semeniperda - Bromus tectorum pathosystem two studies were conducted to address the following: (1) characterization of secondary dormancy in B. tectorum seeds and (2) summer interactions between host and pathogen after summer inoculation. Studies were conducted using dormant and/or non-dormant B. tectorum (along with B. rubens in one study) seeds and two strains of P. semeniperda. Study one used laboratory and field experiments to characterize secondary dormancy in B. tectorum seeds in terms of temperature (0.5-20°C), and water potential (-2.0-0 MPa). Data was used in repeated probit regression analysis to determine hydrothermal parameters (ψb(50), σψb, θHT) for secondary dormancy induction and loss. In the second study seeds were inoculated with one of two strains of P. semeniperda then exposed to intermittent hydration or dry storage at warm temperatures (30-60°C). After treatment seeds were rehydrated and outcomes observed. Optimum conditions for secondary dormancy induction were incubation at -1.0 MPa at 5°C. Seeds were likely to enter secondary dormancy through the cold winter months indicated by an increase or more positive ψb(50), while a decrease or more negative ψb(50) is associated with dormancy loss which is generally observed in the hot, dry summer months. When seeds were inoculated in the summer they only escaped death when summer conditions were ideal for after-ripening which allowed them to germinate rapidly under favorable autumn conditions. However, the pathogen caused high seed mortality no matter the treatment when disease progression advanced enough to inhibit seed germination. Thus this research shows that in areas with frequent summer rain storms, it would be highly advantageous to apply P. semeniperda as a biocontrol on seeds at maturity.

Made available in DSpace on 2016-12-08T16:44:42Z (GMT). No. of bitstreams: 1 PGPV11MA101.pdf: 1302742 bytes, checksum: b7efc06d336ca4a49c0225d1d0b03cfb (MD5) Previous issue date: 2011-02-08 / The objectives of the work were: a) to obtain the damage function equations for multiple pathosystem to calculate the EDT to time fungicides application; b) to quantify the control and damage of the yield components, and c) To relate the brown spot incidence and severity in different growth stages. The experiments were carry out at the NBN Seeds Company during the 2009 and 2010 crop seasons, located in Muitos Capões county, Rio Grande do Sul state, Brazil and in the Agro science Center at Santa Catarina State University only in 2010. The cultivar BRS Cauê was used as susceptible cultivar to brown spot and powdery mildew. The experiments design was in randomized block with four replications. The nine treatments consisting of different rates (half and recommended rates) and fungicide applications number (one, two, three and four) of mixture strobilurin and triazole fungicides, generating the disease gradients intensity. The total area of 5.0 x 2.5 m was the experimental unit in both experiments. The fungicide applications and incidence and severity assessment were done at EC 22, EC 31, EC 39, EC 45 and EC 56 plant development stages. Plants from central rows of each plot were manually harvested and grain yield (GY), one-thousand grain weight (TGW) and granulometry (G) were evaluated. In the first chapter, the damage function equations between disease intensity and GY for each plant development stages, obtained by regression analysis, in both 2009-10 crop growing seasons were significant and negative indicative that increasing disease severity lead into decreasing grain yield. The damage coefficients of these equations can be used to calculate the economic damage threshold. In the second chapter, the values of disease intensity were used to calculate the area under disease progress curve (AUDPC). The GY, TGW, G and AUDPC values were tested using the mean comparison between treatments and the percentage of damage and disease control were determinated. The largest 2009 crop damage was 45.87%, 15.47% and 25.84% for GY, G and TGW, respectively. The ultimate control was 68.11% when considered the severity and four foliar applications, independent of the used dose. In 2010, the greatest damage were 31.16%, 14.02% and 10.76% in Muitos Capões and 39.44%, 23.59% and 45.88%, in Lages, for GY, 10 TGW and G, respectively. The highest percentage of control, based on the leaf severity were 71.63% and 73.96% for Muitos Capões and Lages, respectively. The greater control, independent of used dose were obtained with three and four applications in Muitos Capões and four applications in Lages. In the third chapter, brown spot incidence and severity data were subjected to regression analysis and correlation and the obtained values were significant and positive. The brown spot and powdery mildew diseases incidence and severity recommended by the Technique Indication of crop (TIC) to initiate fungicide applications is 20% and 5% respectively. The severity average values for initiating chemical control are 0.77% and 0.34% respectively for 2009 and 2010 crop seasons when the incidence diseases were substituted in the equations. These values are lower than TIC recommended values / Os objetivos do trabalho foram: a) obter equações de função de dano para patossistema múltiplo para calcular o LDE servindo como critério indicador de aplicação de fungicidas; b) quantificar controle e dano nos componentes de rendimento; e c) relacionar incidência e severidade foliar da mancha marrom em diferentes estádios fenológicos. Os experimentos foram conduzidos nas safras agrícolas 2009 e 2010 na NBN Sementes no município de Muitos Capões, RS; e, na safra de 2010 no Centro de Ciências Agroveterinárias no município de Lages, SC. Em todos os experimentos foi utilizada a cultivar BRS Cauê suscetível à mancha marrom e oídio. O delineamento foi blocos casualisados, com quatro repetições e nove tratamentos constituídos de diferentes doses (meia dose e dose indicada) e número (uma, duas, três e quatro) de aplicações de fungicidas triazóis e estrobilurinas para gerar os gradientes de intensidade das doenças. A área de cada unidade experimental correspondeu a 5,0 x 2,5 m. As aplicações e as avaliações da incidência e severidade foliar ocorreram nos estádios de crescimento (EC) EC 22, EC 31, EC 39, EC 45 e EC 56. A colheita foi manual cortando as plantas das linhas centrais de cada parcela. Foram avaliados rendimento de grãos (RG), massa de mil grãos (MMG) e granulometria (G). No primeiro capítulo, as equações de função de dano entre intensidade de doença e RG para cada EC, obtidas por análise de regressão, em ambas as safras agrícolas, foram significativas e negativas, ou seja, à medida que aumentou a intensidade de doença, diminuiu o RG. Os coeficientes de dano obtidos podem ser utilizados no cálculo do limiar de dano econômico. No segundo capítulo, os valores de intensidade das doenças foram usados para o cálculo da área abaixo da curva de progresso da doença (AACPD). Os valores de RG, MMG, G e AACPD foram submetidos ao teste de comparação de médias. Foram determinados o percentual de dano e de controle das doenças. Na safra 2009, os maiores danos foram 45,87%, 15,47% e 25,84% para RG, MMG e G, respectivamente. O controle máximo foi 68,11% considerando a severidade foliar e quatro aplicações, independente da dose usada. Em 2010, os maiores danos foram 31,16%, 14,02% e 10,76% em Muitos Capões e 39,44%, 23,59% e 8 45,88% em Lages, respectivamente para R, MMG e G. Com base na severidade obteve-se controle de 71,63% e 73,96% em Muitos Capões e Lages, respectivamente. Em Muitos Capões três e quatro aplicações e em Lages quatro aplicações, independente da dose, apresentaram maior percentual de controle. No terceiro capítulo, os dados de incidência e severidade foliar de mancha marrom foram submetidos à análise de regressão e correlação. As equações obtidas foram significativas e positivas. A incidência e a severidade foliar recomendado pela Indicação Técnica da cultura (ITC) para iniciar as aplicações de fungicidas é de 20% e 5%, respectivamente. Substituindo esse valor de incidência nas equações obtidas têm-se valores médios de severidade para iniciar o controle químico de 0,77% e 0,34% respectivamente para 2009 e 2010, inferior ao recomendado pela ITC