Дисертації з теми "Host disease resistance"

Significant differences were observed among 79 geographically diverse Arabidopsis accessions in response to the wheat powdery mildew pathogen, Blumeria graminis f.sp. tritici (Bgt) and the wheat leaf rust pathogen Puccinia triticina (Ptr). In response to Bgt genotypes classified into two major classes based on the degree of compatibility, Wc-1 an accession from Germany expressed significantly high frequency of penetration. Interestingly, in response to Ptr, a high frequency of guard cell death and sub-stomata vesicle formation (SVF) was observed on Wa-1, an accession from Poland. Attempted Ptr infection induced the production of reaction oxygen intermediates (ROI), nitric oxide, salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid and ROI-dependent genes were also detected. Multiple small-to-medium effect quantitative trait loci (QTL) were identified that govern the expression of NMR in Arabidopsis against Ptr. In response to Bgt, a leaf collapse phenotype was observed in Ler when it was pre-treated with Cytochalasin E, an inhibitor of actin microfilament polymerization. Whereas, Col did not express a similar phenotype. This reaction showed a complicated genetic basis with the involvement of several genes. Our genetic analysis revealed two major QTLs on chromosomes one and three with the existence of episatsis effects. A role for ASYMMETRIC LEAVES1 (AS1) in plant immunity has recently been identified. My experiments showed a conserved regulatory function for NSPHAN, an orthologue of ASI gene in Nicotiana sylvestris when challenged with host and nonhost pathogens. This regulatory gene action remained consistent when the as1 mutant was coupled with key Arabidopsis defence related mutants.

The ability to mount an efficient immune response should be an important life-history trait as parasitism can impact upon an individual's fecundity and survival prospects, and hence its fitness. However, immune function is likely to be costly as resources must be divided between many important traits. Whilst many studies have examined host resistance to particular parasite types, fewer have considered general immune responses. Studies that have considered general immune responses tend to do so in vertebrate models. However, the complexity of the vertebrate immune system makes the examination of evolutionary aspects of immune function difficult. Using larvae of the genus Spodoptera (Lepidoptera: Noctuidae) as a model system, this study examines' genetic and phenotypic aspects of innate immunity. The aims were to assess the levels of additive genetic variation maintained in immune traits, to consider possible costs that could maintain this variation, and to assess the role of phenotypic plasticity in ameliorating those costs. A key finding of this study was that high levels of additive genetic variation were maintained in all of the measured Immune traits. Analysis of the genetic correlations between traits revealed potential trade-offs within the immune system and between immune components and body condition. In addition, it was shown that larvae living at high densities invest more in immune function than those living in solitary conditions, suggesting that larvae can minimise the costs of immune function by employing them only when the risk of pathogenesis is high.

In this work we take an evolutionary invasion analysis approach to modelling evolution and use it to describe the selection pressures underlying epidemiological traits in natural host populations harboring endemic infections. Throughout this work a logistic form for host-birth rate allows for disease dependent population dynamics so that the detrimental e ects of infection can be modelled and we also consider the more neglected detrimental e ect whereby infection is linked to infertility. To begin with we give a theoretical introduction to the framework of adaptive dynamics and illustrate it through the established example of the evolution of parasite virulence. We then extend the results to account for condition dependent virulence which is an interaction between host condition (i.e. host stress) and virulence, that has recently generated much attention from empiricists. Many natural systems are seasonal, potentially leading to seasonal stress, and we show how to conduct a study for seasonal host populations and analyse its role in the evolution of density dependent virulence. We then turn our attention to the evolution of resistance beginning with a perspective on the relationship between investment in acquired immunity and the lifespan of hosts and parasites. In our penultimate chapter we derive explicit expressions for optimal investment in the various modes of resistance for a range of epidemiological scenarios. These expressions are then key to understanding our nal chapter where we elaborate further on the established theory by allowing for parasite diversity. The nal chapter highlights the central role played by speci city in the evolution of host defence. Since our approach throughout has been to build complexity onto a baseline model we conclude our discussion with a short section interpreting established results on the coevolution of virulence and resistance from the perspective of our results on the evolution of virulence and resistance.

A pathosystem using Arabidopsis and wheat powdery mildew, Blumeria graminis f. sp. tritici (Bgt), for which Arabidopsis is a non-host, was employed to initiate the genetic dissection of non-host resistance (NHR). The EMS mutagenised population from the Arabidopsis line containing the GST1:LUC transgene, which can facilitate a high throughput mutant screening strategy, have been screened using an ultra low light imaging camera system. Following a mutant screen of approximately 100,000 M2 plants, a number of candidates have been identified that compromise the induction of the LUC transgene in response to attempted Bgt infection. Through this screening, nhr1 was isolated as a putative factor for non-host pathogen recognition. This mutant showed severely compromised GST1 induction and less hypersensitive cell death in response to Bgt inoculation, while exhibiting little difference against other host bacterial and fungal pathogens including Pseudomonas syringae p.v. tomato and Hyaloperonospora parasitica. In addition, nhr1 was sugar dependent in germination. We identified ads3 (ACTIVATED DISEASE SUSCEPTIBILTY 3), an Arabidopsis mutant showing drought resistant as well as disease susceptible phenotype against host and non-host pathogens. ads3 is emf1-D by enhanced expression of Embryonic Flower 1 (EMF1), which had been known as repressor of floral transition in plant. The susceptibility of emf1-D was recapitulated in transgenic Arabidopsis plants ectopically expressing EMF1. Conversely, conditionally decreased EMF1 level in the transgenic plant conveyed disease resistance. emf1-D was drought resistant and hypersensitive to abscisic acid (ABA). We show that ectopic expression of EMF1 modulates a ABA signalling, resulting in susceptibility to pathogens. The other plant link, which is the translationally controlled tumor protein (TCTP) over-expressing line, showed reduced NHR against Bgt. A leucine rich repeat receptor-like kinase (LRK) was isolated as a putative TCTP-interacting protein, and the KO line of the LRK showed less or delayed resistant response to non-host as well as host fungal pathogens. furthermore, the TCTP over-expressing line exhibited hypersensitivity towards ABA. These results suggest that ABA signalling could play a critical role in non-host resistance in plants.

A host can adopt two strategies when facing infection: resistance, where host immune responses prevent or reduce pathogen replication; or tolerance, which refers to all mechanisms that reduce the impact of the infection on host health or performance. Both strategies may be under host genetic control, and could thus be targeted for genetic improvement. Although there is ample evidence of genetic variation in resistance to infection, there is limited evidence to suggest that individuals also differ genetically in tolerance. Furthermore, although resistance and tolerance are typically considered as alternative host defense mechanisms, relatively little is known about the genetic relationship between them and how they change together over time and jointly determine infection outcome. In this thesis, two datasets from experimental challenge infection experiments were considered for investigating tolerance genetics: Porcine Reproductive & Respiratory Syndrome (PRRS), an endemic viral disease which causes loss of growth and mortality in growing pigs; and Listeria monoctyogenes (Lm), a bacterium which causes food-borne infections in mammals. The two datasets differed substantially in size and genetic structure; the PRRS dataset consists of thousands of records from outbred commercial pig populations, whereas the Listeria dataset comprises much fewer records from genetically diverse highly inbred strains of a mice as a model species. The aims of this thesis were to: 1) Identify if genetic variation in host tolerance to infection exists, with case studies in PRRS and listeria, using conventional reaction-norm methodology; 2) Identify if host tolerance, along with resistance, changes longitudinally as infection progresses; 3) Identify whether the WUR genotype is associated with tolerance slope; 4) Analyse the dynamic relationship between host performance and pathogen load over the time-course of infection by examining the relationship at different stages of infection using GWAS; 5) Develop novel trajectory methodology to offer insight into health-infection dynamics, and identify whether there is genetic variation in trajectories; 6) Develop novel trajectory-derived phenotypes that analyse changes in host performance with respect to changes in pathogen load, as an alternative to tolerance, and identify whether genetic variation exists. This study found that conventional reaction-norm methodology is limited to capture genetic variation in tolerance in outbred populations without measures of performance in the absence of infection. However, by utilising repeated longitudinal data on the same dataset, stages of infection (early, mid and late) were defined for each individual, based on host pathogen load. Using these stages of infection, genetic variation in tolerance was identified over all stages of infection and at mid to late stage of infection. Genetic correlation between resistance and tolerance was strong and positive over all stages of infection, and evidence suggested that resistance and tolerance may be under pleiotropic control. Furthermore, this research found that genetic correlations between resistance and growth changed considerably over time, and that individuals who expressed high genetic resistance early in infection tended to grow slower during that time-period, but were more likely to clear the virus by late stage, and thus recover in growth. However, at mid-late stage of infection, those with high virus load also had high growth, indicating potential epidemiological problems with genetic selection of host resilience to infection. Furthermore, genome wide association studies for pathogen load and growth associated with different stages of infection did not identify novel genetic loci associated with these traits than those previously reported for the whole infection period. By adopting conventional methodology, this study found genetic variation in tolerance of genetically diverse mouse strains to Lm and pigs to PRRS, despite statistical problems. The relationship between resistance and tolerance indicated that both traits should be considered in genetic selection programs. By adopting novel trajectory analysis, this study demonstrated that level of expression of resistance and tolerance changed throughout the experimental infection period and, furthermore, that expression of resistance, followed by tolerance, determined survival of infection. Survivors and non-survivors followed different infection trajectories, which were partially determined by genetics. By deriving novel phenotypes from trajectories that explained changes in growth in relation to change in pathogen load at specific time points, and applying these to the PRRS data, this study did not identify genetic variation in these phenotypes. The genetic signal in these phenotypes may have been masked by the fact that individuals were likely at different stages of infection. In summary, this study has shown that genetic improvement of tolerance, in addition to resistance may be desirable, but could be difficult to achieve in practice due to shortcomings in obtaining accurate and unbiased tolerance estimates based on conventional reaction-norms. Infection trajectories have proven to be a promising tool for achieving an optimally timed balance between resistance and tolerance, but further work is needed to incorporate them in genetic improvement programs.

Dieback disease caused by Fusarium and Lasiodiplodia species is a major threat to cocoa production in Ghana and elsewhere in West Africa. Current recommendations involve insecticide application to control mirid bugs whose feeding punctures provide entry points for these fungi. Little is known about the true identity of the causal pathogens of this disease. Earlier work implicated F. decemcellulare as the causal agent and more rarely L. theobromae (Cotterell, 1927; Crowdy, 1947). A total of 117 single spore fungal cultures was established from diseased cocoa stems imported from Ghana. On morphological grounds cultures could be designated as either Fusarium or Lasiodiplodia spp. The Fusarium cultures exhibited inter-isolate variability with respect to macroscopic appearance and macro-conidium morphology, suggesting the presence of more than a single species. The isolates were further characterised by PCR amplification and sequencing of the ITS region of rDNA and comparison with authentic reference cultures. Thirty-seven Fusarium isolates were identified to twenty F. chlamydosporum, nine F. solani and four isolates each of F. oxysporum and F. proliferatum. The thirty-six Lasiodiplodia isolates were identified to two species, twenty-seven L. pseudotheobromae and nine L. theobromae. In pathogenicity tests, F.chlamydosporum, F. oxysporum, F. proliferatum, F. solani and L. pseudotheobromae, previously unknown as pathogens of either cocoa or any member of the Malvaceae, caused significant wilting and dieback in Amelonado seedlings similar to that observed in the field. All isolates exhibited optimal growth at 30 °C on PDA. Disease incidence in 29 and 15 cocoa germplasm lines in the laboratory and greenhouse, respectively, showed reproducible differences in their reaction to necrotic lesion and dieback infection. LCTEEN 37/F was one of the most susceptible genotypes. CATIE1000, T85/799 and MXC 67 were the most tolerant and could be used in cocoa breeding programmes for resistance to dieback.

Background Atlantic salmon (Salmo Salar) is a key aquaculture species in several countries. Since its critical role in economic sector and scientific research, this species has been relatively extensively investigated, in comparison with other farmed and wild aquatic species. However, the genetic components associated with growth and fillet-related traits are lack consistency, and the issue of sea louse disease in both wild and famed salmon is still unsolved. Objectives Overall aim of this project was to understand the genetic basis of growth-related traits and host resistance to sea lice using three large commercial farmed salmon populations. Specifically, the method of quantitative trait loci (QTL) mapping, genome-wide association study (GWAS), and genomic prediction (GS) were utilized to dissect the genetic architectures associated with traits of interest in our experimental populations. Prior to this, linkage mapping was performed to construct a high-density linkage map for Atlantic salmon. Results Linkage map A linkage map was firstly constructed underlying a SNP array containing 132 K validated SNPs. 96,396 SNPs were successfully assigned to 29 chromosomes that correspond to the linkage group number of European Atlantic salmon. 6.5 % of unassigned contigs, which was equal to 1 % of recent whole genome reference assembly (GCA_000233375.4) anchored to exist chromosomes by referring to linkage mapping result. Genetic components associated with growth traits Heritabilities of growth-related traits were about 0.5 to 0.6 in adult and juvenile farmed salmon. The QTL mapping and GWAS suggested the growth-related traits are likely a polygenic genetic architecture with no major QTL segregating. The prediction accuracy estimated by genomic prediction showed that approximately 5,000 SNP markers could achieve the highest accuracy in body weight and length in juvenile salmon within population. Genetic components associated with lice resistance The heritability of lice resistance was 0.22 to 0.33 using pedigree and genetic relationship matrices respectively. GWAS indicated that the host resistance to sea lice was likely polygenic with no individual SNP surpassed the genome-wide significance threshold. Genomic prediction showed that about 5 to 10 K SNPs was able to achieve the asymptote of accuracy in closely related animals, while the greatest advantage of genomic prediction was observed in non-sibling test within population. Conclusions As the growth-related traits and lice resistance are both likely polygenic and population-specific, the genomic prediction is an efficient approach to capture the genetic variances of the traits in selection candidates in experimental population, especially for traits with low heritability such as flesh colour and lice resistance. Family-based selection method is the better choice than mass selection to accumulate the genetic effects in corresponding SNP platform. Given the high cost of genotyping and field data collection, the genotyping-by-sequencing and genotype imputation are likely the way to make significant improvements in relevant research.

Many bacteria, viruses and other parasites cause severe morbidity or mortality in their host populations, creating strong selection for physiological or behavioural mechanisms to avoid disease. Likewise, changes in host susceptibility and contact patterns can dramatically influence the spread of infectious diseases, and hence selection for traits such as virulence and infectivity range in parasites. Understanding how ecological and evolutionary changes in one population affect selection in another represents a key challenge for theoreticians and empiricists alike, and is essential for gaining further insights into host-parasite relationships. This thesis contains theoretical models that explore how genetic and environmental factors shape the evolutionary and coevolutionary dynamics of hosts and parasites. In particular, the roles of genetic specificity (i.e. genotype-by-genotype interactions) and population mixing patterns are investigated, using both mathematical models and computer simulations. A broad range of scenarios are covered, including the coevolution of broad resistance and infectivity ranges (generalism), the persistence of coevolutionary cycling and the maintenance of sex, the effects of mating behaviour on disease prevalence and evolution, and the evolution of sexual and social behaviour. The models presented herein develop our understanding of host-parasite relationships and highlight the importance of genetic interactions and ecological feedbacks.

Host defense peptides (HDPs) provide innate immune defense against invasive S. aureus infection. Recent studies suggest potential cross-resistance between HDPs and the lipopeptide antibiotic, daptomycin (DAP). Isolates that exhibit DAP non-susceptible phenotypes may have virulence advantages and pose challenges to effective treatment. The current studies were performed to compare the efficacies and mechanisms of action of native and engineered HDPs vs. clinical S. aureus strain pairs which differed in susceptibility to daptomycin in vitro. Ultrasensitive radial diffusion and multi-colored flow cytometry were employed to analyze distinctive susceptibilities and mechanisms of resistance, respectively. Overall efficacies were greater vs. DAP-susceptible (DSSA) vs. DAP non-susceptible (DNSA) S. aureus isolates for some but not all HDPs. Efficacy profiles of certain HDPs were influenced by pH, regardless of whether the particular isolate was DSSA or DNSA phenotype. Mechanistically, DSSA and DNSA isolates differed in responses to specific HDPs regarding cell energetics, membrane permeability, cytoplasm membrane turnover, and cell death protease induction. DSSA and DNSA strain pairs exhibited non-identical mechanisms of resistance to HDPs. At pH 7.5, as expected, HDPs hNP-1 and RP-1 exerted significantly greater efficacy on susceptible control strain ISP479C vs. its resistant counterpart ISP479R. These data suggest different mechanisms of HDP resistance are active in differing DNSA strains. These preliminary results are under further investigation, as are the genetic determinant(s) that may emerge during infection. If substantiated, these findings would imply multiple modes of survival of S. aureus in the face of DAP or HDPs.

Plant-based defence mutualisms involve aspects of plant morphology that influence the performance of plant parasites, their natural enemies and trophic interactions. Leaf domatia, small indentations on the underside of leaves, can be structurally complex, and are often inhabited by potentially beneficial mites and other arthropods. Plant morphological traits such as domatia that enhance mutualistic relationships may result in increased plant growth rates, and reproductive success. New Zealand supports ~60 plant species that have domatia, the most speciose genus being Coprosma. The aim of this thesis was to examine factors that affect the production of leaf domatia and their relationship with foliar mite assemblages. The three main objectives of this thesis are: First, to investigate the production of foliar domatia and their susceptibility to limited resources, particularly to carbon availability. Second, to test if domatia are inducible structures during leaf ontogeny in the presence of foliar mites and/or fungi. Finally, to explore the effect of domatia availability on foliar mite assemblages on leaves with and without resident mites. This thesis tested the stated objectives using C. lucida, C. ciliata, C. foetidissima and C. rotundifolia, with a combination of field investigations and controlled manipulative experiments. The cost of domatia production was investigated using two field surveys and two controlled experiments. Under natural conditions the relationship between leaf morphology and domatia were measured in situ and across an altitudinal gradient. The experimental manipulations used carbon and nutrient stress, induced by temperature, light and fertilizer application. The second objective was experimentally tested under field conditions by manipulating foliar mites and fungal densities on C. rotundifolia. The third objective was investigated by manipulating domatia availability on C. lucida shrubs across three different vegetation types. Under field conditions, the number of domatia per leaf was associated with leaf morphology in C. lucida and C. foetidissima, but not C. rotundifolia. Foliar carbon showed a positive, but weak association with domatia production in C. foetidissima and C. ciliata. Altitudinal induced-carbon stress on domatia production was ambiguous. Domatia production in C. foetidissima was positively associated to altitude in field survey (1), and negatively associated in the second survey, with no correlation found between carbon and altitude. Experimental C. rotundifolia shrubs held under elevated night-time temperatures showed a 2.5 fold increase in respiration, a 34% to 91% decrease in daily carbon gain, and 38% decrease in domatia per leaf mass. Domatia production showed no significant differences under nutrient stress. The results showed little evidence to support a role for induction of domatia. Domatia production in new leaves was similar across all experimental treatments. Diverse vegetation types supported 60% higher mite species. Leaves with domatia supported ~22 to 66% higher mite densities, greater colonisation success and more diverse mite assemblages, than those without domatia. In the pastoral vegetation, the absence of predatory mites on experimental shrubs resulted in no differences in fungivorous mite densities regardless of domatia availability. Plant investment in foliar domatia appears associated with the number of available sites on the leaf under field conditions. The role of carbon availability during leaf ontogeny suggests a complex and highly variable association with domatia production. Domatia are constitutive defence structures that influence mite assemblages, mediating both beneficial and antagonistic relationships. This thesis concludes that domatia are in part, carbon-based non-inducible structures that influence mite assemblages, plant-mite and mite-mite interactions, and increase the probability of successful colonisation.

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The morphological and cultural characteristics of G. graminis var. graminis isolates from rice and grasses were studied. The fungus forms two types of mycelia, dark macrohyphae that join laterally to form runner hyphae or rhizomorphs and hyaline or infectious microhyphae, as well as fan shaped hyphae characteristic of the pathogen. Pigmented and lobed hyphopodia on lower leaf sheaths were formed both under natural conditions and artificial inoculations of plants. The perithecia containing asci and ascospores were found on leaf sheaths lesions on field samples. The perithecia were produced on leaf sheaths of inoculated plants as well as on detached sterilized leaf sheaths and on culture medium, potato-dextrose-agar (PDA). Hyphae and hyphopodia were formed from germination tubes of ascospores, and the hyphae under moist stress conditions produced chlamydospores which were initially hyaline and later attained dark color. The culture of Ggg, was characterized by fluffy aerial mycelium, white in the initial stages of growth and later with age, the colony color changed from dirty-white or mouse gray to almost black. The marked diagnostic colony characteristic of whorled appearance was the curling back of marginal hyphae. The amount and time of formation of perithecia varied among the isolates tested. The virulence test conduced with 20 isolates of rice and grasses, showed differences in aggressiveness both on rice seedlings and adult plants. In general, isolates from rice were more aggressive on rice than isolates from grasses. The test with four levels of inoculum (0, 5, 1.0, 2.0, 4.0 g per plant of autoclaved sorghum grains) and two plant ages showed that 60-day old were more susceptible than 35-day old plants. The spontaneous infection of healthy plants was observed in the greenhouse indicating the role of ascospores in the dissemination of black sheath rot in rice. Furthermore, the pathogenicity of ascospores of Ggg on rice plants was confirmed by inoculations tests. Six fields of upland rice were surveyed in the advanced stages of maturation for the incidence of black sheath rot. The disease incidence on tillers, under natural conditions of infection, ranged from 68 to 100%. The pathogenicity of 20 isolates retrieved from rice and grasses were studied. All isolates were pathogenic to rice and grasses such as baranyard grass (Echinochloa crusgalli), fountain grass (Pennisetum setosum) signal grass (Brachiaria sp), crab grass (Digitaria horizontalis), plantain signal grass (Brachiaria plantaginea), indian goose grass (Eleusine indica) and southern sandbur (Cenchrus echinatus). Winter cereals such as wheat, oat, rye, barley and triticale as well as sorghum, corn, and millet exhibited different degrees of susceptibility to the isolate Ggg-a 01. Significant differences were observed in relation to characteristic symptoms on the culm, lesion height, number of tillers or dead plants, presence of characteristic mycelium, fan shaped hyphae, production of hyphopodia and perithecia. The formation of perithecia was not observed on leaf sheaths of inoculated plants of millet, sorghum, southern sandbur and maize. All inoculated wheat plants were killed indicating more susceptibility than other cereals. The resistance of 58 upland rice genotypes were tested in the greenhouse, utilizing rice isolate Ggg-a 01. Of the genotypes assessed, the lesion height of SCIA16 and SCIA08 was significantly shorter compared to the highly susceptible genotype CNAS10351. The progress and dissemination of black sheath rot in rice was studied during two years under field conditions in savanna sensu lato ‘cerrado’. The central line of each plot was inoculated with isolate of Ggg to establish the infection foci. The soil was infested with four levels of inoculum (5.0, 10.0, 20.0 and 40.0 g of autoclaved sorghum grains containing mycelium / 40 cm) and main tiller of plants (4, 8, 16 and 32, tillers per plot/ 40 cm) were inoculated with 2.0 cm-long detached leaf sheaths containing perithecia by insertion between the culm and leaf sheath of the tiller. There was no significant effect of inoculum level on the disease severity obtained by soil infestation with mycelium as well as the plants infected with perithecia. However, the total area under disease progress curve was significantly smaller for plant infection with perithecia than for soil infestation by mycelium, during 2002/2003. The evaluation of disease incidence for the analysis of gradients was based on infected tillers in 1.6 square meter area, five lines on either side of the inoculated 40 cm-long central line. The analysis according models of Gregory (1968) and Kiyosawa & Shiyomi (1972) showed the existence of gradients in the first year, both for levels of inoculum of soil infection by mycelium and plant infection with perithecia. In the second year (2004/2005), there was no well defined gradient for all the treatments. The disease progress was not affected by inoculum levels on soil or plant infections. Monomolecular model was found more adequate in tests conduced under greenhouse conditions while the models of Gompertz and monomolecular, better described the disease progress under field conditions.
Foram estudadas características morfológicas e culturais de isolados de Gaeumannomyces graminis var. graminis provenientes de arroz e capins. O fungo se estabelece formando dois tipos de hifas: macrohifas, escuras, superficiais que se juntam lateralmente e formam cordões ou rizomorfas e microhifas, hialinas ou infecciosas, que penetram no hospedeiro. Forma também hifas em leque sobre as bainhas, a partir de macrohifas, que caracterizam o patógeno. Houve a formação de hifopódios lobados e pigmentados em bainhas, tanto em condições naturais como em inoculações. Observou-se peritécios contendo ascas e ascósporos, característicos do fungo, nas bainhas sobre as lesões em amostras coletadas no campo. Através de inoculação artificial, foram produzidos peritécios em bainhas de plantas, em bainhas destacadas e esterilizadas e em meio de cultura de batata-dextrose-ágar (BDA). Foram formadas hifas e hifopódios a partir de tubos germinativos dos ascósporos e as hifas crescidas em condições de estresse hídrico produziram clamidósporos, inicialmente hialinos e, posteriormente, de coloração escura. O micélio de Ggg, geralmente de aspecto aéreo fofo, é branco no início do crescimento, com variação de cor com a idade, do branco cinza ao marrom oliváceo e quase preto. Uma característica marcante é a aparência espiralada das macrohifas escuras nas bordas da colônia. Entre os isolados testados houve variação na quantidade de peritécios bem como na época de formação. Os testes de virulência realizados com vinte isolados provenientes de arroz e capins apresentaram diferenças em agressividade, tanto em plântulas quanto em plantas de arroz. Em geral, os isolados provenientes de arroz foram mais agressivos em arroz que os isolados de capins. O teste com quatro níveis de inóculo (0,5, 1,0, 2,0, e 4,0 g de inóculo por planta, multiplicado em grãos de sorgo autoclavados) e duas idades de plantas mostrou que as plantas inoculadas aos 60 dias após o plantio foram mais suscetíveis do que aquelas inoculadas aos 35 dias, requerendo menor nível de inóculo para a infecção. A patogenicidade de ascósporos de Ggg em plantas de arroz foi comprovada, bem como o papel dos ascósporos na disseminação do mal-do-pé do arroz. A incidência de mal-do-pé em lavouras de arroz de terras altas nas condições naturais de infecção variou de 68 a 100% de perfilhos infectados, entre seis lavouras avaliadas em fase avançada de maturação. Foi estudada também a patogenicidade dos vinte isolados de Ggg obtidos, provenientes de arroz e capins. Todos os isolados foram patogênicos a arroz e aos capins: capim arroz (Echinochloa crusgalli), capim avião (Pennisetum setosum), capim braquiária (Bachiaria sp.), capim digitaria (Digitaria horizontalis), capim marmelada (Brachiaria plantaginea), capim pé-degalinha (Eleusine indica) e capim timbete (Cenchrus echinatus). Os cereais de inverno, trigo, aveia, centeio, cevada e triticale, bem como sorgo, milho, e milheto apresentaram diferentes graus de suscetibilidade ao isolado Ggg-a 01. As diferenças foram significativas quanto a sintomas típicos na base do colmo, altura de lesão escura na bainha, número de perfilhos ou plantas mortas, presença de micélio característico, hifas em leque e produção de hifopódios e peritécios. Não foram observados peritécios em milheto, sorgo, timbete e milho e a maior suscetibilidade foi apresentada pelo trigo, com a morte de todas as plantas inoculadas. Foi testada a resistência de 58 genótipos de arroz de terras altas, utilizando o isolado Ggg-a 01 proveniente de arroz, em casa-de-vegetação. Entre os genótipos avaliados, SCIA16 e SCIA08 apresentaram altura de lesão significativamente menor, sendo considerados resistentes em relação ao genótipo CNAS10351, altamente suscetível. O progresso e disseminação do maldo- pé do arroz foram estudados durante dois anos, em condições de campo em solo de cerrado. Utilizou-se delineamento experimental de blocos completos ao acaso e quatro repetições. Cada parcela foi constituída de dezenove linhas de sete e cinco metros, respectivamente no primeiro e segundo ano, com espaçamento de quarenta centímetros. Foi inoculada a linha central de cada parcela com isolado de Ggg para estabelecer os focos de disseminação da doença. O solo foi infestado com micélio em quatro níveis de inóculo (5,0, 10,0, 20,0 e 40,0 gramas de grãos de sorgo autoclavados e colonizados com micélio / 40 cm da linha) e perfilhos foram inoculados (4, 8, 16 e 32 perfilhos / 40 cm da linha) com pedaços de bainhas de arroz de dois centímetros de comprimento, contendo peritécios e micélio, inseridos entre o colmo e a bainha. Não houve efeito de níveis de inóculo na severidade da doença, tanto para micélio no solo quanto para peritécios na planta, nos dois anos de experimento. Entretanto, a área total sob a curva de progresso da doença na safra 2002/2003 foi significativamente menor nas plantas infectadas com peritécios, do que nas plantas infectadas através de infestação do solo com micélio. A avaliação de incidência da doença para análise do gradiente foi baseada nos perfilhos contados em 1,6 metros quadrados, compostos de cinco linhas de quarenta centímetros de cada lado da fonte de inóculo, na linha central. A análise de gradiente, conforme modelos de Gregory (1968) e Kiyosawa & Shiyomi (1972) mostrou existência de gradiente no primeiro ano, tanto para níveis de inóculo quanto para os focos provenientes dos dois tipos de inóculo. No segundo ano (2004/2005), não houve gradiente definido para os tratamentos testados. O progresso da doença não foi afetado pelos níveis, tanto na infecção do solo com micélio, quanto na planta com peritécios. Em teste de ajuste de modelo matemático para estudos epidemiológicos, o modelo monomolecular foi o mais apropriado para estudos de mal-do-pé do arroz nas condições de casa-de-vegetação e os modelos de Gompertz e monomolecular são os que melhor descrevem o progresso da doença, nas condições de campo.

The threat from infectious diseases dates as far back as prehistoric times. Pathogens continue to pose serious challenges to human health. The emergence and spread of diseases such as HIV/AIDS, Severe Acute Respiratory Syndrome (SARS), avian influenza, and the threats of bioterrorism have made infectious diseases major public health concerns. Despite many successes in the discovery of anti-infective medications, the treatment of infectious diseases faces serious challenges, which include (i) the emergence and reemergence of infectious pathogens, (ii) the ability of pathogens to adapt and develop resistance to drugs, and (ii) a shortage in the development and discovery of new anti-infective drugs. Host-Oriented Broad-Spectrum (HOBS) treatments have the promising potential to alleviate these problems. The HOBS treatment paradigm focuses on finding drug targets in human host that are simultaneously effective against a wide variety of infectious agents and toxins. In this dissertation, we present a computational approach to predict HOBS treatments by integrative analysis of three types of data, namely, (a) gene expression data representing host responses upon infection by a pathogen, (b) annotations of genes to pre-defined biological pathways and processes, and (iii) genes that are targets of known drugs. Our methods combine gene set-level enrichment with biclustering. We applied our approach to a compendium of gene expression data sets derived from host cells exposed to bacterial or to fungal pathogens, to functional annotation data from multiple databases, and to drug targets from DrugBank. We present putative host drug targets and drugs with extensive support in the literature for their potential to treat multiple bacterial and fungal infections. These results showcase the potential of our computational approach to predict HOBS drug targets that may be effective against two or more pathogens. Our study takes a clean-slate approach that promises to yield unsuspected or unknown associations between pathogens and biological processes, and thus discern candidate gene/proteins to be further probed as HOBS targets. Furthermore, by focusing on host responses to pathogens as captured by transcriptional data, our proposed approach stimulates host-oriented drug target identification, which has potential to alleviate the problem of drug resistance.
Ph. D.

The threat from infectious diseases dates as far back as prehistoric times. Pathogens continue to pose serious challenges to human health. The emergence and spread of diseases such as HIV/AIDS, Severe Acute Respiratory Syndrome (SARS), avian influenza, and the threats of bioterrorism have made infectious diseases major public health concerns. Despite many successes in the discovery of anti-infective medications, the treatment of infectious diseases faces serious challenges, which include (i) the emergence and reemergence of infectious pathogens, (ii) the ability of pathogens to adapt and develop resistance to drugs, and (ii) a shortage in the development and discovery of new anti-infective drugs. Host-Oriented Broad-Spectrum (HOBS) treatments have the promising potential to alleviate these problems. The HOBS treatment paradigm focuses on finding drug targets in human host that are simultaneously effective against a wide variety of infectious agents and toxins. In this dissertation, we present a computational approach to predict HOBS treatments by integrative analysis of three types of data, namely, (a) gene expression data representing host responses upon infection by a pathogen, (b) annotations of genes to pre-defined biological pathways and processes, and (iii) genes that are targets of known drugs. Our methods combine gene set-level enrichment with biclustering. We applied our approach to a compendium of gene expression data sets derived from host cells exposed to bacterial or to fungal pathogens, to functional annotation data from multiple databases, and to drug targets from DrugBank. We present putative host drug targets and drugs with extensive support in the literature for their potential to treat multiple bacterial and fungal infections. These results showcase the potential of our computational approach to predict HOBS drug targets that may be effective against two or more pathogens. Our study takes a clean-slate approach that promises to yield unsuspected or unknown associations between pathogens and biological processes, and thus discern candidate gene/proteins to be further probed as HOBS targets. Furthermore, by focusing on host responses to pathogens as captured by transcriptional data, our proposed approach stimulates host-oriented drug target identification, which has potential to alleviate the problem of drug resistance.
Ph. D.

Erythrocytes infected by mature forms of the Plasmodium falciparum parasite adhere to other components of the vascular space, a behavior considered critical to the pathogenesis of severe malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a highly variant antigen expressed by the parasite and subject to switching during the course of an infection. The host ligands of PfEMP1 include CD36, ICAM-1 and the ABO antigens. By employing a series of population- and family-based association studies from multiple African populations, we examined whether variation in the genes underlying these molecules affects susceptibility to severe malaria. Our results suggest that a common frameshift mutation in the ABO glycosyltransferase, responsible for blood group O, is associated with protection from severe malarial phenotypes (P=2x10⁻⁷), particularly severe malarial anaemia. However, we found no significant disease associations with variation in either the ICAM1 or CD36 genes. We focused on two particular functional polymorphisms, the missense ICAM-1Kilifi and the CD36 nonsense mutation T1264G. We genotyped both markers in around 10,000 individuals, but neither demonstrated an association with severe malarial phenotypes. Malaria has been a profound selection pressure shaping human genetic diversity. The last decade has seen the development of several haplotype-based methods to detect signatures of recent positive evolutionary selection. These techniques are potentially invaluable tools in our hunt for genetic variants that protect from life threatening malaria. We used simulations and empirical data from the International HapMap Project to demonstrate the validity of searching for long regions of haplotype homozygosity, as an approach to finding alleles undergoing selective sweeps. We analysed genetic data from a range of populations, particularly those utilized by HapMap, to investigate whether our candidate genes were associated with signals of recent positive selection. We characterized the distribution of a selection event associated with the CD36 1264G allele, focused in Central-West Africa, and demonstrated a novel signal of low population differentiation at the ABO gene, suggestive of longstanding balancing selection. Our work confirms that variation in the host ligands of PfEMP1 modulates severe malaria susceptibility, and highlights the value of using signals of selection, along with functional experiments and genetic association studies, to dissect the biology of severe malaria.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

Host-plant resistance is an important component of integrated pest management. Host resistance to phytophagous insects and mites based on chemical and morphological characteristics has been identified in various agronomic crops. The present study was undertaken to examine how variation in selected morphological and chemical characteristics of beach strawberry Fragaria chiloensis (L.) Duchesne affects population parameters of the twospotted spider mite Tetranychus urticae Koch. The results from this study show that clones of F. chiloensis and the cultivar "Totem" vary in suitability for the twospotted spider mite. Selected clones of F. chiloensis and the cultivar "Totem" differed also in the following foliar characteristics: densities of glandular and nonglandular trichomes and concentrations of total and catechol-based phenolics. In contrast, qualitative analysis of phenolic compounds, determined via HPLC, revealed a close similarity between F. chiloensis and F. x ananassa (cultivated strawberry) and among selected clones of F. chiloensis. Mite oviposition was negatively correlated with densities of glandular and nonglandular trichomes and concentrations of total foliar phenolics. In spite of these significant relationships, mite oviposition was best predicted by a combination of interacting plant characteristics rather than by any individual characteristic. Entrapment by sticky exudates from glandular trichomes appears to explain the negative relationship between mite survival and density of glandular trichomes. Spider mite development was negatively related to concentrations of foliar phenolics; twospotted spider mite development is delayed on leaves with high concentrations of phenolics. The correlation between mite development and foliar phenolics was greater when catechol-based phenolics were used.
Land and Food Systems, Faculty of
Graduate

Les chloroplastes des feuilles de betteraves rhizomaniees sont appauvries en pigments, en proteines, en liquides polaires et leur activite photosynthetique est reduite. Dans les cellules virosees, l'etude ultrastructure montre une association des amas de virus avec le reticulum endoplasmique granuleux. Un protocole pour la preparation de suspensions enrichies en cytosores isoles de polymyxa betae est propose. Le champignon est retrouve a tous les niveaux du sol de quatre parcelles rhizomaniees ou saines de la region de pithiviers. Une terre rhizomaniee reste infectieuse aprese un an et demi de lagune en bassin

En élevage porcin, le sevrage est une période critique caractérisée par un stress nutritionnel, environnemental et social, avec une forte sensibilité des animaux à la diarrhée. Le microbiote intestinal doit s'adapter à un changement alimentaire, avec le passage d'une alimentation lactée à un aliment plus complexe à base de céréales, et les animaux sont soumis à la pression exercée par les agents infectieux environnementaux. Les bactéries entérotoxiques Escherichia coli (ETEC) sont les principaux agents pathogènes responsables de la diarrhée post-sevrage et peuvent entrainer des pertes économiques considérables. Le rôle de la génétique de l’hôte dans la sensibilité à l'infection est bien établi, le polymorphisme des gènes Mucine 4 (MUC4) et Fucosyltransférase 1 (FUT1) étant associé à la sensibilité à ETEC F4 et F18, respectivement. Nous avons réalisé deux études afin d’analyser l’effet de facteurs pouvant influer sur la sensibilité des porcelets à la diarrhée au sevrage. Dans une première étude, nous avons évalué l'impact de l'âge au sevrage sur la diversification du microbiote intestinal, par comparaison du microbiote d’animaux sevrés à différents âges. Quarante-huit porcelets de race Large White ont été répartis en quatre groupes de 12 animaux sevrés à 14 jours (sevrage précoce), 21 ou 28 jours (âge au sevrage courant en élevage intensif) et 42 jours (sevrage tardif). La composition bactérienne du microbiote a été établie par séquençage du gène de l'ARNr 16S d’ADN fécal extrait de selles prélevées le jour du sevrage, sept jours après et à l'âge de 60 jours. Nous avons montré que le sevrage tardif augmente la diversité du microbiote, avec une plus grande abondance de Faecalibacterium prausnitzii identifiée comme bénéfique chez l'homme. Ces résultats suggèrent que la composition du microbiote intestinal pré-sevrage conférée par un sevrage à 42 jours pourrait améliorer la santé intestinale des porcelets, en leur permettant d’acquérir un microbiote plus diversifié avec des bactéries potentiellement bénéfiques lors du sevrage. La seconde étude a eu comme objectif d’évaluer, chez des porcelets sevrés, les effets du génotype des gènes MUC4 et FUT1 et des voies d'administration de l’amoxicilline sur la présence de diarrhée et la composition du microbiote fécal, lors d'une infection naturelle par des souches d'ETEC multirésistantes. Soixante et onze porcelets ont été répartis en trois groupes: deux groupes se différenciant par la voie d'administration de l'amoxicilline, parentérale (P) ou orale (O), et un groupe témoin sans antibiotiques (C). Nous avons confirmé que MUC4 et FUT1 sont des marqueurs génétiques de l’hôte pour la sensibilité aux infections à ETEC et montré que le traitement à l'amoxicilline pouvait avoir des effets néfastes sur la santé du porc au cours d'une infection à ETEC multirésistante, accentués lors d’une administration par voie orale. Les deux études ont mis en évidence l’importance de considérer des méthodes alternatives de conduite d’élevage. Avec la nécessité de limiter l'utilisation d'antibiotiques, la sélection de génotypes résistants, la supplémentation en next-generation probiotics dans l’alimentation et une meilleure optimisation de l'âge au sevrage devraient être prises en compte dans les pratiques, afin de favoriser un microbiote intestinal diversifié, capable de réduire les diarrhées au sevrage
In pig production systems, weaning is a crucial period characterized by nutritional, environmental and social stress. During this process, piglets are susceptible to diarrhoea and the gut ecosystem needs to adapt to dietary changes, from a milk-based diet to a solid and more complex cereal-based feed, and to environmental pathogen pressure. One of the most important etiological agent of the post-weaning diarrhoea (PWD) is the Enterotoxigenic Escherichia coli (ETEC) able to cause severe outcomes and considerable economic losses to farmers worldwide. A role of host genetics in infection appearance is well-established, the SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes being associated with the susceptibility to ETEC F4 and ETEC F18, respectively.To investigate aspects related to weaning diarrhoea, two studies have been performed. The aim of the first study was to evaluate the impact of weaning age on gut microbiota diversification in piglets comparing animals at different weaning ages. Forty-eight Large White piglets were divided into four groups of 12 animals weaned at 14 days old (early weaning), 21 or 28 days old (main weaning ages in pig intensive farming) and 42 days old (late weaning). In each group, faecal bacteria composition was assessed by sequencing the 16S rRNA gene of faecal DNA on the weaning day, 7 days post-weaning and at 60 days of age. Our results showed that late weaning increases the gut microbiota diversity including a higher abundance of Faecalibacterium prausnitzii, reported as beneficial in humans. Our results suggest than the pre-weaning gut microbiota composition conferred by a late weaning at 42 days of age could enhance gut health in piglets. This would provide a competitive advantage to piglets accumulating a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning transition.The aim of the second study was to evaluate the effects of the host-genotype and different routes of amoxicillin administration on the presence of diarrhoea and the microbiota composition, during a natural infection by multi-resistant ETEC strains in weaned piglets. For this purpose, seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes – parenteral (P) or oral (O) and a control group without antibiotics (C). Our results confirmed the MUC4 and FUT1 as host genetic markers for the susceptibility to ETEC infections. Moreover, our data highlighted that amoxicillin treatment may produce adverse outcomes on pig health in course of multi-resistant ETEC infection and this effect is stronger when the antibiotic is orally administered than parenterally.Both studies highlighted the importance of alternative control measures related to farm management in controlling weaning related diarrhoea. With a need to limit the use of antibiotics, selection of resistant genotypes, next-generation probiotics supplementation in feed, and correct procedures of weaning age, should be considered in farm management practices in order to preserve a balanced and stable gut microbiota and consequently reduce occurrence of diarrhoea at weaning
Lo svezzamento rappresenta un momento cruciale nell’allevamento suinicolo ed è caratterizzato da stress nutrizionale, ambientale e sociale. In questa fase, i suinetti risultano a maggior rischio di insorgenza di diarrea in quanto la microflora intestinale deve adattarsi ai cambiamenti alimentari legati al passaggio da una dieta a base lattea ad un alimento solido a base di cereali e più complesso e all’elevata pressione infettiva ambientale. Uno dei più importanti agenti eziologici responsabili della diarrea post-svezzamento (PWD) è Escherichia coli Enterotossigeno (ETEC) in grado di provocare gravi quadri clinici nonché ingenti perdite economiche per gli allevatori. Che ci sia una componente genetica nell'evoluzione di queste infezioni è stato ben definito attraverso l’individuazione degli SNP situati sui geni Mucine 4 (MUC4) e Fucosyltransferase 1 (FUT1) associati rispettivamente alla suscettibilità nei confronti di ETEC F4 e ETEC F18. Nella presente tesi sono illustrati due studi che hanno avuto l’obiettivo di approfondire alcuni aspetti legati alla comparsa di diarrea durante lo svezzamento. Lo scopo del primo studio è stato quello di valutare l'impatto dell'età di svezzamento sulla diversità del microbiota intestinale, confrontandone la composizione in suinetti svezzati a diverse età. Quarantotto suinetti di razza Large-White sono stati suddivisi in quattro gruppi da 12 soggetti, svezzati rispettivamente a 14 giorni di età (svezzamento precoce), a 21 o 28 giorni (età di svezzamento principale nell'allevamento intensivo) e a 42 giorni (svezzamento tardivo). In ogni gruppo è stata valutata la composizione batterica fecale il giorno dello svezzamento, 7 giorni post-svezzamento e a 60 giorni di età, sequenziando il gene 16S rRNA dal DNA batterico fecale. I risultati ottenuti hanno evidenziato come lo svezzamento tardivo aumenti il grado di diversificazione del microbiota intestinale, aumentando l’abbondanza di Faecalibacterium prausnitzii, già considerato benefico per l'uomo. Emerge, inoltre, come la composizione del microbiota intestinale nel pre-svezzamento associata allo svezzamento tardivo incrementi il livello di salute intestinale nei suinetti. Tale condizione, comporterebbe un notevole vantaggio per gli animali che acquisiscono una maggiore differenziazione del microbiota intestinale, incrementando l’abbondanza di batteri beneficiali prima di affrontare lo stress dello svezzamento. Lo scopo del secondo studio è stato quello di valutare gli effetti del genotipo dell’ospite e le vie di somministrazione dell’amoxicillina sulla comparsa della diarrea e sulla composizione del microbiota intestinale, durante un'infezione naturale causata da ETEC multi-resistente, in suinetti svezzati. A tale scopo, settantuno suinetti sono stati divisi in tre gruppi: due gruppi diversificati dalla via di somministrazione dell’amoxicillina - parenterale (P) o orale (O), e un terzo gruppo di controllo in cui non sono stati somministrati antibiotici (C). I risultati ottenuti hanno confermato il ruolo di MUC4 e FUT1 quali marcatori genetici di suscettibilità alle infezioni da ETEC. Inoltre, i nostri dati hanno evidenziato come la somministrazione di amoxicillina possa influenzare negativamente lo stato di salute dei suini in corso di infezione da ETEC, effetti ancora più evidenti quando la somministrazione antibiotica avviene per via orale. Entrambi gli studi hanno sottolineato l'importanza di adottare misure alternative legate al management aziendale per il controllo della diarrea post-svezzamento. Nell’ottica di limitare l'utilizzo di antibiotici, azioni quali la selezione di genotipi resistenti, l'integrazione di probiotici di nuova generazione nei mangimi ed una corretta gestione dell’età di svezzamento, dovrebbero essere prese in considerazione nelle pratiche gestionali aziendali al fine di preservare un microbiota intestinale equilibrato e stabile e di conseguenza ridurre l'insorgenza di diarrea allo svezzamento

[ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales.
[CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals.
[EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections.
The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM).
Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035
TESIS

Mycosphaerella graminicola (anamorph Septoria tritici) causes Septoria tritici blotch, a globally important disease of winter wheat. Resistance and pathogenicity generally vary quantitatively. The pathogen reproduces both sexually and asexually, and the pathogen population is highly genetically variable. Several unresolved questions about the epidemiology of this pathosystem are addressed by this research. Among them are whether cultivar-isolate specificity exists, how partial host resistance affects pathogen aggressiveness and sexual reproduction, and how host genotype mixtures influence epidemic progression and pathogenicity. At its release in 1992, the cultivar Gene was highly resistant to M. graminicola, but that resistance had substantially dissolved by 1995. Six of seven isolates collected in 1997 from field plots of Gene were virulent to Gene seedlings in the greenhouse, while 14 of 15 isolates collected from two other cultivars were avirulent to Gene. Gene apparently selected for strains of M. graminicola with specific virulence to it. In a two-year experiment, isolates were collected early and late in the growing season from field plots of three moderately resistant and three susceptible cultivars, and tested on seedlings of the same cultivars in the greenhouse. Isolates were also collected from plots of two susceptible cultivars sprayed with a fungicide to suppress epidemic development. Isolate populations were more aggressive when derived from moderately resistant than from susceptible cultivars, and more aggressive from fungicide-sprayed plots than from unsprayed plots of the same cultivars. Over 5,000 fruiting bodies were collected in three years from replicated field plots of eight cultivars with different levels of resistance. The fruiting bodies were identified as M. graminicola ascocarps or pycnidia, or other. In all three years, the frequency of ascocarps was positively correlated with cultivar susceptibility, as measured by area under the disease progress curve, and was also positively associated with epidemic intensity. For three years, four 1:1 mixtures of a moderately resistant and a susceptible wheat cultivar were planted in replicated field plots. Isolates from the plots were inoculated as bulked populations on greenhouse-grown seedlings of the same four cultivars. Mixture effects on disease progression varied among the years, and were moderately correlated with mixture effects on pathogenicity.
Graduation date: 2002

"August 2004"
Bibliography: leaves 146-169.
vi, 169 leaves : ill, (some col.), photos (col.) ; 30 cm.
Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2004

Grapevine powdery mildew is caused by Erysiphe necator, an Ascomycete fungus and an obligate biotroph restricted to growth on its grapevine host. Biotrophic pathogens form a stable association with host cells without directly causing cell death, and take up nutrients from, in the case of powdery mildew ( PM ), host epidermal cells ( Rumbolz et al., 2000 ). As the fungus grows, its increasing biomass becomes a strong nutrient sink capable of altering assimilate flow and storage in the host. To identify host genes that may mediate nutrient delivery to powdery mildew infected tissues and therefore may contribute to disease susceptibility, a candidate gene approach using degenerate and RT - PCR, and a nontargeted approach using microarray analysis was instigated. Once identified, " susceptibility genes " could be targeted for manipulation to provide alternative resistance strategies based on reduced susceptibility in the future. In addition to genes encoding pathogenesis and stress related proteins, microarray analysis revealed that transcript levels of a putative metal transporter and a cell wall structural protein were elevated in infected berry skin, while aquaporin water channels and genes associated with photosynthesis were generally repressed. Degenerate PCR was used to isolated new cell wall invertase, monosaccharide and amino acid transporter genes and initial RT - PCR revealed that expression of genes involved in sugar mobilisation were the most significantly modulated by powdery mildew infection. Previously unreported hexose transporters ( HTs ), ( VvHT3, VvHT4 and VvHT5 ) and a cwINV ( VvcwINV ) had been isolated from cDNA prepared from powdery mildew infected grapevine leaves. Full length clones of grapevine HTs and cwINV were obtained by RACE PCR. Heterologous expression of the three new HTs in yeast confirmed that VvHT4 and VvHT5 mediated glucose uptake, while VvHT3 did not function in the yeast system. However, transient expression of a translational fusion of the VvHT3 protein with green florescence protein in onion epidermal cells indicated that it is targeted to the plasma membrane of plant cells. Quantitative RT - PCR analysis of these new genes, together with previously reported grapevine HTs and cytoplasmic and vacuolar invertases, indicated that expression of VvcwINV and VvHT5, were significantly up - regulated by PM infection, while a vacuolar invertase was strongly down - regulated by PM infection. Invertase activity assays were in agreement with these findings, showing elevated sucrolytic activity in insoluble fractions and reduced sucrolytic activity in soluble fractions. These results suggest that apoplasmic phloem unloading of sucrose in the infected leaf is elevated and that VvHT5 is induced to recover the additional hexoses from the apoplasm. Basic localisation studies indicated that VvHT5 and VvcwINV are not induced specifically in powdery mildew infected leaf regions, but are induced in a more diffuse distribution within infected leaves. To determine if induction of VvHT5 and VvcwINV is specific to PM infection or if other stimuli may also mediate these responses, leaves were inoculated with downy mildew or stressed by wounding. Transcript levels of VvHT5 and VvcwINV were elevated by wounding and downy mildew infection, suggesting that the induction of these genes may be part of a general stress response. To explore the signalling pathways that may underlie these responses, leaves were treated with the plant growth regulators ethylene, jasmonate and abscisic acid. Exogenous application of ethylene and methyl jasmonate only marginally affected the expression of the genes studied, however foliar application of abscisic acid ( ABA ) induced gene expression changes similar to those observed in response to powdery mildew infection and wounding. Promoter sequences of VvHT3, VvHT4, VvHT5 and VvcwINV were isolated and analysed for the presence of regulatory elements. Compared with the promoters of VvHTs that were not induced by pathogen infection or wounding, the VvHT5 and VvcwINV promoters contained numerous motifs associated with induction by ABA including ABRE, Myc and Myb binding elements. The path of sugar loading into the mesocarp of grape berries during ripening is still poorly understood and few molecular components associated with this process have been described. Quantitative RT - PCR was used to monitor the expression of five HTs and VvcwINV during Cabernet sauvignon and Shiraz berry development and ripening. Of the three new HTs reported here, the expression of VvHT3 is most consistent with a potential role in sugar loading, while VvHT5 is induced late in this process. VvcwINV transcript levels were high pre - ripening and also during the later stages of ripening, therefore based on this expression pattern, a role for this enzyme during ripening is not clearly evident. These results are discussed in terms of an apoplasmic step in phloem unloading in ripening grape berries. This study has provided new insights into the molecular and biochemical processes associated with the formation of carbohydrate sink metabolism in response to stress stimuli, and sugar delivery to grape berries during ripening. ABA - dependant pathways may mediate the stress - associated induction of VvcwINV and VvHT5, presumably to recruit additional carbohydrates to the affected organ to energise repair and defence responses. At this stage it is unknown if this response is beneficial to pathogen nutrition, however potentially, modification of genes associated with carbohydrate sink metabolism could provide an alternative way to engineer resistance to this pathogen.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2006.

碩士
國立臺灣大學
植物病理與微生物學研究所
107
Bakanae or foolish seedling disease of rice is caused by the ascomycetous fungus Fusarium fujikuroi, of which is widespread in many rice growing areas. Current protection strategies rely on the fungicides, but this results in chemical-resistant F. fujikuroi and other undesirable environmental effects. An alternative approach involving RNAi, termed host-induced gene silencing (HIGS), of which generating siRNA molecules in plants that target to the specific mRNAs of pathogens and thereby resulted in their degradation. Calcineurin belongs to a Ca2+/calmodulin-dependent phosphatase, which is comprised of heterodimer with a catalytic (Cna1) and a regulatory (Cnb1) subunits. There have been known that calcineurin mediates Ca2+ signaling that regulates the growth, stress responses and virulence in fungal kingdom. Based on pharmacological inhibition of calcineurin in F. fujikuroi, we found that calcineurin inhibitor FK506 or cyclosporinA can strongly inhibit the growth of F. fujikuroi. We therefore aim to determine whether repressing the expression of CNA1 or CNB1 gene in F. fujikuroi by the HIGS strategy in rice can enhance resistance against bakanae disease. By using Agrobacterium-mediated gene transformation strategy, the FfCNA1-Ri and FfCNB1-Ri constructs have been introduced into rice plants, respectively, and the copy number of transgenes were analyzed by Southern blot hybridization in the transgenic lines. Based on the pathogen inoculation assay and fungal biomass quantification, results showed that the transgenic rice plants that carry the FfCNA1-Ri or FfCNB1-Ri constructs increased resistance against bakanae disease, promising its potential use in the future.

The effects of diurnal temperature fluctuation and the utility of degree days for modeling clubroot on canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin were assessed using microscopy and qPCR, and in field trials. Temperature fluctuation had little effect on pathogen development. The optimal temperature for root hair infection was 25° C. Air and soil degree days and rainfall were used as metrics for estimating clubroot development, with only limited success. Several cultivars of cabbage (Brassica oleracea L. var. capitata) with unknown clubroot resistance mechanism(s) were assessed using staining and microscopy, and qPCR. In field trials, ‘Bronco’ was susceptible to clubroot (100 DSI), ‘Kilaherb’ was resistant (0 DSI), and ‘B-2819’ was intermediate (53 DSI). Plasmodiophora brassicae was present in cortical tissue of all cultivars. A delayed disease phenotype in ‘B-2819’ may indicate a quantitative resistance genotype that could be exploited in research on resistance genes and breeding.