Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis. / Ph. D.

Chronic exposure to the ubiqutious airborne fungus, Alternaria alternata, has long been implicated in the development and exacerbation of human allergy and asthma. Alt a 1 was identified previously by several groups as the major allergen secreted by A. alternata, due to its IgE-specific reactivity with sera from atopic patients. Despite the well-documented clinical importance of Alternaria and its major allergen, little knowledge exists regarding their role and interaction with the innate immune system. Here for the first time we characterize the innate immune response to A.alternata and verify the significance of Alt a 1 in contributing to this response in human airway cells and murine models. Our studies establish a baseline response for both a chronic and single-challenge murine inhalation model with Alternaria spores. Both models demonstrate live conidia induce a robust response, arguably more pathologically relevant compared to studies employing Alternaria extracts. We also elucidate the overall importance of Alt a 1 by utilizing recombinant Alt a 1 protein, A. alternata (Δalt a 1) deletion mutants, and an A.alternata (Alt a 1+) overexpression mutant. Both Alt a 1 protein and A. alternata conidia stimulated production of pro-inflammatory cytokines/chemokines in mice after a single intranasal challenge. Infiltration of effector cells (macrophages, neutrophils, eosinophils, and lymphocytes) into the lungs along with other hallmarks of airway inflammation was observed. In addition, Alt a 1 protein and conidia evoked secretion of pro-inflammatory cytokines in treated human airway epithelial cells while the Alt a 1+ overxpression mutant induced a significantly higher response. In contrast, spores of Δalt a 1 caused an attenuated response in both human cells and murine lungs suggesting that this single protein may play a major role in inducing the innate immune response in airway epithelium at the organismal level. Finally, we identified key biochemical properties of the Alt a 1 protein including a single histidine required for esterase activity and a unique RXLR-like motif which controls Alt a 1's ability to bind external lipids and enter human airway cells. Overall, these results improve our understanding of how Alternaria induces innate immunity and identifies possible therapeutic targets within allergenic proteins. / Ph. D.

Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x ¿ 2x) or interploid crosses (2x ¿ 4x and 4x ¿ 2x). In 2x ¿ 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x ¿ 4x and 4x ¿ 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x ¿ 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in `Fortune¿ mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced gamete formation to other citrus genotypes besides clementines and `Fortune¿ mandarin, a maximum likelihood method based on parental heterozygosity restitution of centromeric loci was developed and successfully applied in sixteen mandarin cultivars. The new method developed in the study allows inferring the restitution mechanism both at population level and even at individual level. Maternal origin of 2n gametes was confirmed for all triploid hybrids and SDR was proposed as the restitution mechanism for all analyzed progenies. The information acquired from the mode of heterozygosity restitution in citrus was useful to determine the genetic and phenotypic structures of new triploid populations arising from different breeding strategies. We studied these structures for the resistance to Alternaria brown spot (ABS), a serious fungal disease producing necrotic lesions on fruits and young leaves in susceptible citrus genotypes. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from SDR to map a genome region linked to ABS resistance in triploid citrus progeny. The monolocus dominant inheritance of the susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. A 3.3 Mb genomic region linked to ABS resistance was located near the centromere on chromosome III, which includes clusters of resistance genes. SSR and SNP markers were developed for an efficient early selection of ABS resistant hybrids and they are currently used in our breeding program to perform marker assisted selection. The knowledge obtained in this thesis on the mechanism of sexual polyploidization and inheritance of concrete traits in citrus will allow implementing much more efficient triploid breeding programs on the basis of current and future needs. Indeed, applied outcomes of this PhD are already routinely used in the IVIA triploid breeding program. / Cuenca Ibáñez, J. (2013). Mechanisms of sexual polyploidization and inheritance in triploid citrus populations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34671 / Premios Extraordinarios de tesis doctorales

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science / Alternaria alternata, is an airborne ubiquitous fungus, which is associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. While dendritic cells, which are known as the classical antigen presenting cells, play an important function in antigen recognition at the early stage of immune response and then pass the signal to other immune cells. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. As the results in this study, we found that BMDCs were highly sensitive to A. alternata spores and their cell wall components. Following stimulation with these molecules, the expression of major histocompatibility complex (MHC) molecules and other co-stimulators, were highly up regulated. In order to determine how different cell wall components affect other immune cells through BMDCs, we conducted co-culture experiments of BMDCs and different T cells population in this study. Both spores and cell wall component did not induce IL-4, a featured Th2 cytokine, in those co-culture studies. Interestingly, we found that cell wall component induced the switching of the T helper cell population to the Th17 type. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways.

Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P). / Ph. D. / Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and allergy to several fungal species including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying how the allergen, Alt a 1, from the fungus, <i>Alternaria alternata</i>, can cause an allergic response in the human airways. In several studies, 90-100% of allergic patients who have allergy to Alternaria, have proteins in their bloodstream that specifically recognize Alt a 1. This indicates that they are allergic to Alt a 1. Though we know that these patients have allergy to Alt a 1, we do not know how this protein causes the characteristic symptoms of allergy, such as a runny nose, watery eyes, hives, and breathing difficulty. Published studies have increasingly made it clear that molecules on the surface of cells that line the airways are important players in the body’s response to allergens. A dust mite allergen, Der p 2, can interact with one such receptor on human cells. The receptor may not be the only way that allergens can cause a response. Studies have shown that allergens can directly enter human cells. For example, a Birch tree allergen, Bet v 1, has been shown to enter human lung cells. This project was focused on two primary aims; (1) Identify how human airway cells response to Alt a 1, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to cause human airway cells to produce several molecules that lead to the characteristic symptoms of allergy, and that this response was dependent on a receptor on human airway cells. We also found that Alt a 1 rapidly enters human airway cells.

Die pilzlichen Erreger der Tomate Alternaria solani und des Manioks Colletotrichum gloeosporioides f.sp. manihotis verursachen große Ertragsausfälle in Westafrika. In der vorliegenden Arbeit wurden die Blattkrankheiten nach Applikationen mit wässerigen Extrakten aus Westafrika (Ausgangsmaterial: organischer Haushaltsmüll) unter kontrollierten Bedingungen unterdrückt. Im Pathosystem Tomate - A. solani erzielten die Extrakte eine Befallsverminderung um bis zu 94 % im Vergleich zur Kontrollvariante und im Maniok - C. gloeosporioides wurde die Anzahl der gebildeten Acervuli/cm² Blattoberfläche bis um 87 % reduziert. Der primäre Wirkmechanismus der Extrakte gegen die Pathogene ist Antagonismus, hervorgerufen von den Mikroorganismen in den Extrakten. Isolierte Bakterien aus den Extrakten hemmten das Wachstum der Pathogene in Plattentests, was auf die Bildung von fungistatischen Substanzen zurückzuführen war. Die Bakterien, die aus den Extrakten isoliert und identifiziert wurden, gehörten überwiegend zu den Bakteriengattungen Pseudomonas und Bacillus. Die Extraktionsbedingungen nahmen in Hinblick auf die Wirksamkeit der Extrakte gegen die Pathogene eine wichtige Stellung ein. Die Untersuchungen zeigten einen Zusammenhang zwischen gestiegener Mikroorganismenanzahl und Befallsreduktion. Durch die Auswahl optimaler Extraktionsbedingungen erhöhte sich die Anzahl der Koloniedichte in den Extrakten; dies führte zur einer stärkeren Befallsreduzierung an der Tomate und am Maniok. Hierbei waren die Umgebungstemperatur und die Häufigkeit des Durchmischens des Kompost-Wasser-Gemisches von besonderer Bedeutung. Extrakte, die während der Extraktionszeit einer Temperatur von 20°C ausgesetzt waren, erzielten eine stärkere Befallsreduzierung von A. solani an der Tomate und wiesen eine höhere Mikroorganismendichte auf als Extrakte, die einer Temperatur von 30°C und 35°C ausgesetzt waren. Tägliches Durchmischen des Wasser-Kompost-Gemisches während der Extraktionszeit erhöhte die Mikroorganismendichte um das Siebenfache in den Extrakten und die Wirksamkeit gegen A. solani und C. gloeosporioides im Vergleich zu den Extrakten, die nur zu Beginn durchmischt wurden. Die Lagerungszeit der fertigen Komposte hatte einen Einfluß auf die Mikroorganismendichte der wässerigen Extrakte sowie auf deren Wirksamkeit. Extrakte, die aus frischen Komposten hergestellt wurden, zeigten einen besseren suppressiven Effekt gegen die Pathogene und wiesen eine höhere Mikroorganismenanzahl auf als Extrakte, deren Komposte schon länger gelagert worden waren. Die Anreicherung der Extrakte mit dem Formulierungshilfsstoff Xanthan steigerte die Wirksamkeit der Extrakte gegen A. solani an Tomatenpflanzen und gegen C. gloeosporioides f.sp. manihotis an abgetrennten Maniokblättern. Angereicherte Extrakte reduzierten den Befall um bis zu 46 % mehr als der Extrakt ohne Formulierungshilfsstoffe, während die Steigerung der Wirksamkeit nach Anreicherung mit Alginat oder Zellulose eine Abhängigkeit von der Herkunft der Komposte zeigte. Eine erhöhte ß-1,3-Glucanase-Aktivität wurde am vierten Tag nach der Applikation von Extrakten an der Tomate und an der Batate festgestellt. Diese Ergebnisse belegen die Bedeutung von Spritzbehandlungen mit wässerigen Kompostextrakten gegen die Pathogene A. solani und C. gloeosporioides f.sp. manihotis und stellen eine erfolgversprechende Alternative zur Bekämpfung von Pilzkrankheiten in Westafrika dar. / Great yield losses of cassava in West Africa are caused by fungal pathogens that also affect tomato, e.g. Alternaria solani and Colletotrichum gloeosporioides f sp. manihotis. In the present study these foliar diseases were effectively suppressed under controlled conditions after application of watery compost extracts to tomato and cassava The disease incidence of A. solani on tomato was reduced up to 94 % and the number of the acervuli produced on the surface of cassava leaves was reduced up to 87% compared to the control treatment. Reduction is due to antagonistic effects of the microorganisms in the extracts towards the pathogens. Bacterial strains isolated from the extracts suppressed the growth of the pathogens in dual-culture-tests due to the production of antibiotics. Bacteria isolated and identified from the extracts were mostly pseudomonas and bacillus strains. The conditions used for extraction of compost played a significant role for their effectiveness against different pathogens. The investigations showed that optimisation of extract conditions increased the density of microorganisms in the extracts and led to a higher disease reduction on tomato and cassava plants. Temperature and stirring frequency of the compost - water mixture during the extraction period were very important factors for increased effectiveness. Extracts incubated at 20 °C during the extraction period caused a higher disease reduction on tomato and showed a higher density of microorganisms, than extracts that were incubated at 30 or 35 °C. Daily stirring of the compost - water mixture during the extraction period increased the density of microorganisms in the extracts sevenfold and increased the effectiveness against A. solani and C. gloeosporioides compared to the extract that was stirred only once at the beginning. The Effectiveness as well as the density of the microorganisms were depended on the incubation time of the compost extracts. Extracts produced from fresh composts showed greater suppressive effects against pathogens and higher densities of microorganisms than extracts of composts that were stored for a longer time. Enrichment of the extracts with the formulate substance xanthan increased the effectiveness against A. solani on tomato plants and against C. gloeosporioides on detached leaves of cassava and reduced the disease incidence up to 46% compared to the extract without formulate substances. The increase of the effectiveness after enriching with alginate was depended on the origin of the compost. An increased ß-1,3- glucanase activity were determined on tomato and sweet potato leaves 4 days after the application of the extracts onto the plants. The results of this study show that watery compost extracts can be successfully applied against the cassava and tomato pathogens A. solani and C. gloeosporioides f. sp. manihotis and offers easily applicable methods for the reduction of fungal diseases in West Africa.

Made available in DSpace on 2015-03-26T13:37:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4434052 bytes, checksum: e6bd53f46965b44a3dcdbb9a5a63b4ff (MD5) Previous issue date: 2008-02-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work aimed to select tomato prokaryotic phylloplane residents for biocontrol purposes, by using Xanthomonas campestris pv. vesicatoria and Alternaria solani as challenging pathogens. Additionally, to perform a screening test using detached leaves. Consequently, leaves from healthy tomato plants were collected at Viçosa, MG and epiphytic prokaryote populations were extracted by shacking them in PBS (0,85% NaCl in 0,1M phosphate buffer saline, pH 7.0) following sonication (60Hz, 20 minutes) and serial dilution and plating in culture medium. Three hundred bacterial isolates were obtained and preserved in refrigerator, with periodical tubetube transfer, in a refrigerator. The screening was performed in two selection cycles. In the first selection in greenhouse, with three replicates per isolate and Xanthomonas campestris pv. vesicatoria as challenging pathogen, 79 isolates were able to reduce disease by 82%. In a second selection cycle, using 6 replicates per isolate, the 33 isolates were able to reduce severity by 50% in average. The 33 isolates had their antagonistic potential tested against the tomato fungal and bacterial pathogens Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani and Corynespora cassiicola in vitro conditions. Out of the 33 isolates, the isolate RFK-24 inhibited growth of Xathomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis and Alternaria solani while isolate RFS-183 was able to inhibit Alternaria solani, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, but none of them inhibited Corynespora cassiicola. Being RFK-24 and RFS- 183 the ones with a wider antagonistic potential out of the 33 previously selected, they were chosen for continuing the research with detached leaves and the challenging pathogens Alternaria solani e Xanthomonas campestris pv. vesicatoria. There was a positive correlation between disease severity reduction in plants the greenhouse and in detached leaves, for both antagonists. For situations involving large number of isolates to undergo mass screening, the approach with detached organs may replace the laborious and time consuming greenhouse screening. / O presente trabalho teve como objetivo isolar procariotos residentes de filoplano de tomateiro como agentes de biocontrole contra o patógeno Xanthomonas campestris pv. vesicatoria. 300 isolados foram obtidos a partir de folhas sadias de tomateiros coletadas na micro-região de Viçosa-MG, as quais foram submetidas à extração das populações procarióticas epifíticas em solução de tampão fosfato (PBS) com emprego de ultra-som (60Hz, 20 min.) e semeadura de diluições em série em placas de Petri contendo meio 523. Em casa-de-vegetação, numa primeira etapa 79 isolados apresentaram capacidade de reduzir, em média, 82 % da severidade de doença. Desses, numa segunda etapa, 33 isolados apresentaram redução média da severidade de doença em 50 %. Os 33 isolados foram submetidos a testes de antibiose in vitro, em que foi avaliada a potencialidade em inibir o crescimento bacteriano e fúngico dos seguintes patógenos do tomateiro: Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani e Corynespora cassiicola. Dos 33 isolados pré- selecionados verificou-se que o isolado RFK-24 foi capaz de inibir o crescimento dos patógenos, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis e Alternaria solani e o RFS-183 em inibir o crescimento dos patógenos Alternaria solani, Xanthomons campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, mas ambos não inibiram o crescimento da Corynespora cassiicola. Os dois isolados foram selecionados para ensaios in vivo e em folíolos destacados, contra os patógenos desafiantes Alternaria solani e Xanthomons campestris pv. vesicatoria. Para ambos foi observada a correlação entre a severidade de doença apresentada em casa-de-vegetação e os resultados em folíolos destacados, como a inibição da germinação de conídios do patógeno fúngico e a supressividade em meio semi-seletivo dos antagonistas sob o patógeno bacteriano. Os resultados obtidos com o método com folíolos destacados e sua correlação com os dados de biocontrole experimental em casa-de-vegetação permitem deduzir que o uso de folíolos destacados pode ser utilizado como método para seleção massal.

La carotte présente un grand intérêt nutritionnel comme source alimentaire en caroténoïdes. Pourtant, la connaissance des mécanismes d’accumulation de ces composés est un enjeu majeur. Si le déterminisme génétique a été relativement bien étudié, l’impact de stress sur l’accumulation des caroténoïdes chez la carotte reste méconnu. Ce travail de thèse vise donc à déterminer (i) l’impact de stress biotique et abiotique appliqués individuellement ou en combinaison sur les teneurs en caroténoïdes dans les feuilles et racines de carotte,(ii) les mécanismes de régulation pouvant expliquer ces variations (iii) si le métabolisme secondaire est affecté spécifiquement, indépendamment du métabolisme primaire.Les résultats mettent en évidence un effet négatif des différentes conditions de stress, en particulier appliquées en combinaison, sur les teneurs en caroténoïdes dans les feuilles et les racines de carotte, mais dépendent du génotype. La régulation transcriptionnelle au niveau de la voie de biosynthèse des caroténoïdes ne peut expliquer qu’en partie les variations de teneurs. Les variations de teneurs en chlorophylles des feuilles et sucres des racines sont corrélées à celles des teneurs en caroténoïdes, suggérant des mécanismes communs de régulation.Ce travail montre que l’impact de stress en culture, et en particulier leur combinaison, est une composante importante de l’élaboration de la qualité nutritionnelle. Les travaux doivent être poursuivis afin d’établir un schéma plus précis de la régulation de l’accumulation des caroténoïdes chez la carotte. / Carrot presents a high nutritional interest as a carotenoid intake source. However, knowledge about accumulation mechanisms of these compounds is a major issue. While genetic determinism was relatively well studied, the impact of stresses on carotenoid accumulation in carrot remains unknown. This thesis work aims to determine (i) the impact of biotic and abiotic stresses applied individually or in combination on carotenoid contents in carrot leaves and roots, (ii) the regulation mechanisms which could explainthese variations and (iii) if secondary metabolism is specifically affected independently from primarymetabolism. Results bring to light a negative effect of the different stress conditions, particularly applied in combination, on carotenoid contents in carrot leaves and roots but depend on genotypes. Transcriptional regulation based on carotenoid biosynthetic genes can only partially explain contentvariations. Chlorophyll content variations in leaves and sugar content variations in roots are correlated to those of carotenoids suggesting common regulation mechanisms. This work shows that the impact of stress on culture, and particularly in combination, is an important determinism of nutritional quality. Further works need to be performed to establish a more precise regulation network pattern of carotenoid accumulation in carrot.

In rural inland, south-eastern Australia, allergy to the fungus Alternaria is prevalent and an important risk factor for asthma. The aim of the thesis was to investigate the distribution and factors influencing allergens of Alternaria in the air. As airborne allergenic spores were thought to arise from harvesting of nearby crops, two towns with different agricultural practices were studied. Moree has two crop harvesting periods in summer and autumn whilst Wagga Wagga has one harvesting period in summer. Over two years, air was sampled daily in Wagga Wagga and Moree using Burkard traps. The reliability of measurements from a single site to represent the distribution of airborne concentrations of spores across each town was examined using data from three traps simultaneously, sited 2.0 to 4.9 km apart, over four weeks. Substantial intra-class correlation coefficients (ICC) were observed between the three sampling sites across both towns (ICC=0.52, 95% CI 0.30-0.71 to 0.76, 95% CI 0.61-0.87) when counts of Alternaria spores were relatively high. The correlation was poor when counts were low. Of more than 365 trap tapes examined, the two microscopic traverses strongly correlated for counts of Alternaria spores (ICC=0.95, 95% CI 0.94-0.96). Alternaria was detected in both towns throughout the two year period with peaks in spore concentrations reflecting the season of crop harvesting in each region. Individual exposure to spores was examined. Thirty three subjects (adults and children from nine families) wore nasal air samplers and personal air samplers both inside and outside their homes. The effects of activity, location, age on the inhalation of Alternaria spores and variation between individuals in the same environment were determined. Every subject inhaled Alternaria spores. Personal exposure to Alternaria in the home environment varied substantially between subjects. Levels of fungal spores inhaled were higher during periods of activity than during rest, and higher while subjects were outdoors than indoors. During outdoor activity, the number of Alternaria spores inhaled ranged from 4 to 794 (median 11) spores/hr. Sources of airborne spores was investigated by sampling air above wheat and cotton crops near the towns during harvesting and non-harvesting periods, in a grain and cotton seed storage shed, and a cotton gin. Substantially higher concentrations were detected above crops during harvesting periods compared to non-harvesting periods. Peaks were associated with harvesting and other activities where plants were manipulated. By regression analysis spore concentrations in both towns were modelled against those detected above crops and with weather variables. Only one crop sampling period (cotton harvest) independently correlated with concentrations in town. Analysis combining all data showed concentrations of spores above crops correlated with spore concentrations in the town when lagged by one day. Variables of rainfall and maximum temperature influenced concentrations in both towns, and wind direction in Wagga Wagga alone. Parents of asthmatic children were asked by questionnaire in which locations symptoms were provoked. Asthma was reported to be exacerbated at grain farms and with disturbance of local vegetation in town and home gardens. Nasal sampling confirmed that activities that disturbed dust or vegetation increased the inhalation of spores. The factors that release allergen from spores were determined in a modified Halogen immunoassay. Approximately 60% of spores released allergen, and the proportion was influenced by isolate, nutrient availability, viability, and not influenced by sunlight or culture age up to 21 days. Germinating the spores significantly increased the proportion that released total allergen and Alt a 1 (p<0.0001). Alt a 1 appears to be a minor contributor to the total allergen released from spores except when spores have germinated. Conclusions: People living in inland rural regions of Australia are exposed to substantial quantities of allergenic spores of Alternaria. Exposure is a highly personal event and is largely determined by disturbance of local vegetation releasing spores such as from nearby crops by wind, harvesting, slashing, transport and processing of produce, and from within town and home gardens. Most spores inhaled are likely to be allergenic, with potency potentially increasing with viability.

La carotte, légume racine le plus consommé au monde, voit sa production fortement impactée par diverses maladies dont la brûlure foliaire. Cette maladie, provoquée par le champignon nécrotrophe Alternaria dauci, est considérée comme la plus préjudiciable des maladies foliaires de la carotte. Développer des variétés présentant un niveau de résistance fort et durable à cette maladie est l'un des objectifs principaux des sélectionneurs, notamment en cumulant différentes résistances partielles dans un même génotype. Afin d'optimiser un tel cumul, il est nécessaire de comprendre les mécanismes associés à ces résistances. Dans la première partie de cette thèse, nous avons étudié l'implication de métabolites de défense produits par la carotte dans la résistance partielle de la plante à A. dauci. Nous avons montré un effet inhibiteur de la 6- méthoxymelléine (6-MM) et du falcarindiol, sur le développement du champignon. Cet effet est plus important avec le falcarindiol. De plus, la teneur en falcarindiol est supérieure chez le génotype résistant (Boléro) en comparaison avec le génotype sensible (Presto) et, suite à l'inoculation de la plante par A. dauci, la 6-MM s'accumule de façon plus importante dans les feuilles du génotype résistant. Ces molécules de défense pourraient ralentir le développement du champignon et participer ainsi à la résistance. Dans une seconde partie, nous nous sommes intéressés à l'effet de métabolites toxiques produits par le champignon sur des cultures cellulaires de carotte. Nos résultats montrent une corrélation entre le comportement des cellules traitées (viabilité cellulaire, embryogenèse somatique et activité enzymatique) et la sensibilité/résistance des génotypes évaluée au niveau de la plante entière. Parmi les génotypes les plus résistants au champignon, le génotype I2 résiste mieux à l'application d'extraits fongiques que le génotype K3. Ainsi, la résistance partielle de la carotte face à A. dauci semble inclure des résistances aux toxines. Dans le dernier chapitre, nous avons recherché un lien entre la résistance partielle et les mécanismes de défense de la plante, en particulier ceux liés à la voie des jasmonates. Nos premiers résultats montrent la surexpression du gène PR4 chez le génotype résistant K3, comparativement aux autres génotypes, y compris I2. Nous avons par ailleurs recherché des sites polymorphes (SNP) dans les séquences de quatre gènes de défense. Deux gènes (PAL, GLP) présentent un polymorphisme de séquence entre génotypes. Dans le cas de GLP, les SNPs mis en évidence permettent de différencier un génotype sensible (H1) du génotype K3. L'ensemble de ces résultats semble indiquer la présence d'une diversité des modalités de la résistance partielle de la carotte face à A. dauci. Ainsi, la résistance aux toxines semble jouer un rôle plus important chez I2 que chez K3. Inversement, la résistance de K3, et non celle de I2, semble impliquer la voie des jasmonates. Audelà de leur application en amélioration, ces travaux jettent une lueur sur les mécanismes de la résistance partielle, mécanismes encore très peu connus, même parmi les interactions modèles.