La maîtrise du développement des moisissures retrouvées dans le contexte agro-alimentaire parmi les flores microbiennes d'altération ou technologiques répond à des enjeux économiques et sanitaires importants. Le développement des moisissures peut être affecté par des facteurs abiotiques comme la température, l'activité de l'eau (aw) ou la composition gazeuse. L'évaluation de l'effet de ces facteurs via des outils de mycologie prévisionnelle vise à prévoir l'altération fongique des aliments. Ce travail a pour objectif d'explorer l'impact des conditions environnementales sur la physiologie de moisissures d'intérêt pour l'industrie agro-alimentaire.L'effet de la température, de l'aw ajustée par du glycérol ou du chlorure de sodium (NaCl) du pH et de la composition gazeuse a été évalué sur la germination des spores et/ou la croissance radiale de cinq moisissures: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum et Penicillium roqueforti. Au niveau appliqué, ces travaux ont montré que l'effet du NaCl ou de la composition gazeuse peuvent être inclus dans une approche de mycologie prévisionnelle. Le choix des souches représentatives d'une espèce fongique et l'état physiologique des spores utilisées comme inoculum ont un impact significatif sur les modèles prédictifs.Au niveau fondamental, des marqueurs ont été recherchés pour évaluer l'effet des facteurs abiotiques sur la physiologie des spores. La température et l'aw ont un effet significatif sur l'état physiologique des spores et leur germination. La recherche de marqueurs moléculaire, contribuera aux connaissances de l'effet des facteurs abiotiques sur la physiologie des moisissures. / In the food processing industry, controlling the development of filamentous fungi encountered as spoilers or technological cultures address significant economic and sanitary issues. Fungal development in foods is mainly determined by abiotic factors including temperature, water activity (aw) or the headspace gas composition. The quantification of these respective effects through a predictive mycology approach aims at preventing fungal food spoilage. The present work aims at exploring the effect of environmental conditions on the physiology of filamentous fungi of interest in the food processing industry.The effect of temperature, aw (adjusted with glycerol of sodium chloride), pH and headspace gas composition was evaluated on conidial germination and/or radial growth of five fungi isolated from dairy products: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti. The present work suggests that the specific effects of sodium chloride or gas composition could be included in predictive mycology approaches. It was also demonstrated that the selection of strains representative of a fungal species and the physiological state of conidia utilized as inoculum have a significant effect on the final predictive models.At the fundamental level, markers were investigated to study the effect of abitoic factors on the physiological state of spores. The temperature and aw significantly affected the physiological state of spores and their germination kinetics. The investigation of markers at the molecular level could provide better knowledges on the effect of abiotic factors on the physiology of filamentous fungi.

Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.

A resposta imune adaptativa do tipo Th1 é necessária para proteção contra P. Brasiliensis. Sabendo que células dendrítica são APCs eficientes na ativação da resposta imune mediada por células, investigamos o potencial dessas células em iniciar a resposta imune inata em camundongos suscetíveis (B10.A) e resistentes (A/J) a PCM. Inicialmente, observamos que células dendríticas pulmonares de camundongos B10.A são mais fagocíticas quando comparadas com células de camundongos A/J. Além disso, observamos que a fagocitose na presença de laminarina foi inibida somente em células dendríticas pulmonares de animais B10.A. A produção de citocinas por células dendríticas pulmonares de camundongos A/J mostrou baixa concentração de IL-10, IL-12 e TNF-α. Ao contrário, células dendríticas pulmonares de camundongos B10.A produziram altas concentrações de TNF-α e IL-10, mas, a produção de IL-10 foi significativamente inibida na presença de laminarina. Nós também observamos que células dendríticas pulmonares de camundongos TLR-2KO foram deficientes na produção de IL-10. Além disso, a expressão gênica para TLR-2 aumentou após infecção em camundongos B10.A, mas não nos A/J. Posteriormente, observamos que a capacidade de células dendríticas pulmonares de camundongos suscetíveis em induzir ativação de células T foi diminuída. De acordo com nossos resultados, sugerimos que P. brasiliensis induz células dendríticas regulatórias em camundongos suscetíveis, os quais promovem a produção de IL-10, contribuindo para a suscetibilidade de camundongos B10.A contra a infecção por P. brasiliensis. / An adaptive Th1-type immune response is required for protection against P.brasiliensis. Knowing that DC are the most effective APCs for inducing cellmediated immune responses, it is thus important to investigate lung DC and their potential to initiate an immune response in mice susceptible and resistant to PCM. Initially, we observed that lung DC from susceptible mice were more phagocytic than cells from resistant mice and we observed that phagocytosis in the presence of laminarin was inhibited only in DC from susceptible mice. Cytokines produced by DC from resistant mice showed a low concentration of IL-10, IL-12 and TNF-α. In contrast, DC from susceptible mice produced a high concentration of TNF-α and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DC from TLR-2KO mice presented a defective production of IL-10. We found that the gene expression for TLR2 is increased after infection in B10.A, but not in A/J mice. Thus, the capacity of lung DC from susceptible mice in inducing T cell activation was decreased. In conclusion, our data suggest that P.brasiliensis induces regulatory DC in susceptible mice, which promotes IL-10 production contributing to the susceptibility of B10.A mice against P.brasiliensis infection.

The overall aim of this research was to improve the understanding of the ecology and diversity of Trichoderma species within the soil and rhizosphere of onion (Allium cepa L.) and potato (Solanum tuberosum L.) under intensive management in New Zealand. The indigenous Trichoderma population was measured in a field trial at Pukekohe over a three year period under six different crop rotation treatments. The treatments included two continuous onion and potato rotations (intensive), two onion/potato mixed rotation (conventional), and two green manure rotations (sustainable). Results showed that Trichoderma populations were stable in both the rhizosphere and bulk soil (1.5 x 10² to 8.5 x 10³ CFU g⁻¹ ODS). The planting and incorporation of an oat (Avena sativa L.) green manure in the sustainable rotations positively increased Trichoderma colony forming unit (CFU) numbers in the rhizosphere soil from 3.4 x 10² to 2.5 x 10³ g⁻¹ ODS. A Trichoderma species identification method was developed based on colony morphology. Representative isolates were verified using restriction fragment length polymorphism (RFLP) and DNA sequencing. The method allowed for rapid and reliable identification of isolated Trichoderma species. Five species were identified in the Pukekohe soil: T. asperellum, T. atroviride, T. hamatum, T. harzianum and T. koningii. Results showed identical species diversity between the rhizosphere, rhizoplane and bulk soil. The species did not strongly compete between each other for the rhizosphere ecological niche and differences in species proportions seemed to be caused by environmental factors rather than the rotation treatments. The incorporation of oat green manure in pots did not significantly promote the indigenous Trichoderma population size and diversity in the rhizosphere of onion plants up to 4 months old. The identified species were the same as in the field trial. The incorporation of onion scale residues was shown to result in low Trichoderma and high Penicillium CFU numbers and a reduction in plant size. Additionally, the presence of high levels (6.0 x 10⁵ CFU g⁻¹ ODS) of Penicillium CFU was negatively correlated with the presence of Trichoderma CFU. The effect of oat incorporation on Trichoderma saprophytic growth was also investigated in a soil sandwich assay and revealed no significant differences. A series of experiments indicated that onion extract obtained from dry onion scale residues had no antifungal activity against either Trichoderma or Penicillium and instead tended to promote their hyphal growth and sporulation. It also showed that competition between Penicillium and Trichoderma isolates was limited despite the ability of Penicillium to produce a wide range of inhibitory substances. Four indigenous Trichoderma species (T. atroviride, T. hamatum, T. harzianum and T. koningii) were shown to be rhizosphere competent in a split tube experiment over a 6 week period. The results of this experiment revealed that, the Trichoderma species clearly displayed differences in their ability to colonise the rhizosphere of young onion seedlings. Species such as T. koningii had the greatest rhizosphere colonising ability regardless of soil depth while T. harzianum displayed the weakest ability. Results also indicated that when inoculated as a mixture the four species competed with one another to colonise the rhizosphere. Overall, this research indicated that the studied crop rotation treatments and the use of oat as a green manure did not strongly promote indigenous Trichoderma populations. Species diversity was constant throughout the research with T. hamatum and T. koningii being the most frequently isolated species.

A resposta imune adaptativa do tipo Th1 é necessária para proteção contra P. Brasiliensis. Sabendo que células dendrítica são APCs eficientes na ativação da resposta imune mediada por células, investigamos o potencial dessas células em iniciar a resposta imune inata em camundongos suscetíveis (B10.A) e resistentes (A/J) a PCM. Inicialmente, observamos que células dendríticas pulmonares de camundongos B10.A são mais fagocíticas quando comparadas com células de camundongos A/J. Além disso, observamos que a fagocitose na presença de laminarina foi inibida somente em células dendríticas pulmonares de animais B10.A. A produção de citocinas por células dendríticas pulmonares de camundongos A/J mostrou baixa concentração de IL-10, IL-12 e TNF-α. Ao contrário, células dendríticas pulmonares de camundongos B10.A produziram altas concentrações de TNF-α e IL-10, mas, a produção de IL-10 foi significativamente inibida na presença de laminarina. Nós também observamos que células dendríticas pulmonares de camundongos TLR-2KO foram deficientes na produção de IL-10. Além disso, a expressão gênica para TLR-2 aumentou após infecção em camundongos B10.A, mas não nos A/J. Posteriormente, observamos que a capacidade de células dendríticas pulmonares de camundongos suscetíveis em induzir ativação de células T foi diminuída. De acordo com nossos resultados, sugerimos que P. brasiliensis induz células dendríticas regulatórias em camundongos suscetíveis, os quais promovem a produção de IL-10, contribuindo para a suscetibilidade de camundongos B10.A contra a infecção por P. brasiliensis. / An adaptive Th1-type immune response is required for protection against P.brasiliensis. Knowing that DC are the most effective APCs for inducing cellmediated immune responses, it is thus important to investigate lung DC and their potential to initiate an immune response in mice susceptible and resistant to PCM. Initially, we observed that lung DC from susceptible mice were more phagocytic than cells from resistant mice and we observed that phagocytosis in the presence of laminarin was inhibited only in DC from susceptible mice. Cytokines produced by DC from resistant mice showed a low concentration of IL-10, IL-12 and TNF-α. In contrast, DC from susceptible mice produced a high concentration of TNF-α and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DC from TLR-2KO mice presented a defective production of IL-10. We found that the gene expression for TLR2 is increased after infection in B10.A, but not in A/J mice. Thus, the capacity of lung DC from susceptible mice in inducing T cell activation was decreased. In conclusion, our data suggest that P.brasiliensis induces regulatory DC in susceptible mice, which promotes IL-10 production contributing to the susceptibility of B10.A mice against P.brasiliensis infection.

<p>Eastern black walnut (<i>Juglans nigra</i> L.) ranks among the most highly valued timber species in the central hardwood forest and across the world. This valuable tree fills a critical role in native ecosystems as a mast bearing pioneer on mesic sites. Along with other <i>Juglans</i> spp. (Juglandaceae), <i>J. nigra</i> is threatened by thousand cankers disease (TCD), an insect-vectored disease first described in 2009. TCD is caused by the bark beetle <i>Pityophthorus juglandis</i> Blackman (Corthylini) and the phytopathogenic fungus <i>Geosmithia morbida</i> Kol. Free. Ut. & Tiss. (Bionectriaceae). Together, the <i>P. juglandis</i>-<i>G. morbida</i> complex has expanded from its historical range in southwest North America throughout the western United States (U.S.) and Europe. This range expansion has led to widespread mortality among naïve hosts <i>J. nigra</i> and <i>J. regia</i> planted outside their native distributions.</p> <p> The severity of TCD was previously observed to be highest in urban and plantation environments and outside of the host native range. Therefore, the objective of this work was to provide information on biotic and abiotic environmental factors that influence the severity and impact of TCD across the native and non-native range of <i>J. nigra</i> and across different climatic and management regimes. This knowledge would enable a better assessment of the risk posed by TCD and a basis for developing management activities that impart resilience to natural systems. Through a series of greenhouse-, laboratory- and field-based experiments, environmental factors that affect the pathogenicity and/or survival of <i>G. morbida</i> in <i>J. nigra</i> were identified, with a focus on the microbiome, climate, and opportunistic pathogens. A number of potentially important interactions among host, vector, pathogen and the rest of the holobiont of TCD were characterized. The <i>holobiont</i> is defined as the whole multitrophic community of organisms—including <i>J. nigra</i>, microinvertebrates, fungi and bacteria—that interact with one another and with the host.</p> <p>Our findings indicate that interactions among host, vector, pathogen, secondary pathogens, novel microbial communities, and novel abiotic environments modulate the severity of TCD in native, non-native, and managed and unmanaged contexts. Prevailing climatic conditions favor reproduction and spread of <i>G. morbida</i> in the western United States due to the effect of wood moisture content on fungal competition. The microbiome of soils, roots, and stems of trees and seedlings grown outside the host native range harbor distinct, lower-diversity communities of bacteria and fungi compared to the native range, including different communities of beneficial or pathogenic functional groups of fungi. The pathogen <i>G. morbida</i> was also associated with a distinct community of microbes in stems compared to <i>G. morbida</i>-negative trees. The soil microbiome from intensively-managed plantations facilitated positive feedback between <i>G. morbida</i> and a disease-promomting endophytic <i>Fusarium solani</i> species complex sp. in roots of <i>J. nigra</i> seedlings. Finally, the nematode species <i>Bursaphelenchus juglandis</i> associated with <i>P. juglandis</i> synergizes with <i>G. morbida</i> to cause foliar symptoms in seedlings in a shadehouse; conversely, experiments and observations indicated that the nematode species <i>Panagrolaimus</i> sp. and cf. <i>Ektaphelenchus</i> sp. could suppress WTB populations and/or TCD outbreaks.</p> <p>In conclusion, the composition, function, and interactions within the <i>P. juglandis</i> and <i>J. nigra</i> holobiont play important roles in the TCD pathosystem. Managers and conservationists should be aware that novel associations outside the host native range, or in monocultures, intensive nursery production, and urban and low-humidity environments may favor progression of the disease through the effects of associated phytobiomes, nematodes, and climatic conditions on disease etiology. Trees in higher diversity, less intensively managed growing environments within their native range may be more resilient to disease. Moreover, expatriated, susceptible host species (<i>i.e.</i>, <i>J. nigra</i>) growing in environments that are favorable to novel pests or pest complexes (<i>i.e.</i>, the western U.S.) may provide connectivity between emergent forest health threats (<i>i.e.</i>, TCD) and native host populations (<i>i.e.</i>, <i>J. nigra</i> in its native range).</p>