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Impactos ambientais podem interferir nas características da biomassa microbiana, no processo de ciclagem de nutrientes, nas características físicoquímicas e também na diversidade da microbiota e da macrofauna. O objetivo desse trabalho foi conhecer as diferentes interações entre estes atributos e identificar os indicadores da qualidade do solo envolvidos com o tempo de recuperação. Três áreas com estágios diferentes de recuperação (5, 10 e 20 anos) foram comparadas com uma floresta Estacional Semidecídua nativa (NT) com intuito de estudar o comportamento da microbiota, da macrofauna e de suas interações com os atributos físico-químicos. Foram coletadas amostras em 15 pontos por áreas, escolhidos aleatoriamente. Dentre os atributos microbiológicos, a maior atividade das enzimas urease, fosfatase ácida e desidrogeanse foi encontrada na área nativa. O mesmo foi constatado para a respiração basal e para o carbono e nitrogênio da biomassa microbiana. A análise da estrutura da comunidade de Bacteria, feita a partir de TRFLP, separou as áreas, nativa e de 20 anos de recuperação, das demais, somente no verão. A densidade do solo, a umidade e a microporosidade afetaram negativamente os indicadores microbiológicos do solo, sendo em conjunto com o carbono total do solo os principais fatores discriminantes das áreas. Ocorreu maior presença das espécies de FMA A. spinosa, A. colossica, A. lacunosa, G. decipiens e Gigaspora sp. na área NT e das espécies G. viscosum, A. mellea, A. scrobiculata e S. heterogama na área R05 e G. rosea nas áreas R10 e R20. As principais variáveis ambientais que explicam a relação com as espécies de FMA foram microporosidade, macroporosidade, atividade da fosfatase ácida e densidade, umidade, CBM, NBM e N-NO3. Efeito sazonal sobre as espécies de FMA também foi observado. Maiores valores de proteína do solo relacionada com glomalina (GRSP) foram encontrados no inverno e somente a proteína do solo relacionada com glomalina facilmente extraível (EE-GRSP) separou a área NT das demais. Observou-se, também, alta correlação dessa glicoproteina com os atributos físico-químicos e microbiológicos. Em relação à fauna edáfica ocorreu efeito da sazonalidade e do método de coleta utilizado (armadilhas e monolito). Houve diferença entre os índices de Shanon, Simpson e Pielou somente na época seca, sendo as maiores diferenças nas áreas mais antigas (NT e R20). Maior riqueza foi encontrada ao se utilizar o método de monolito. De acordo com a análise discriminante, Diplopoda foi o principal grupo para as duas épocas e os dois métodos de coleta. Porosidade, densidade basal, umidade, nitrogênio total do solo e atividade das enzimas desidrogenase e urease foram fatores importantes para a separação dos grupos da fauna no gradiente de recuperação ambiental. Esse trabalho mostrou que os atributos biológicos e físicoquímicos de qualidade do solo interagem e se modificam de acordo com a idade das áreas e com a sazonalidade. / Environmental impactation can affect microbial biomass, nutrient cycling, processes, physical-chemical characteristics and also the diversity of microbes and edaphic fauna. The aim of this study was to understand the different interactions between these attributes and to identify the indicators of soil quality involved in the recovery process. Three areas with different stages of recovery [5 (R05), 10 (R10) and 20 (R20) years] were compared with a native semideciduous forest (NT) in order to study the behavior of microbes, macrofauna and their interactions with the physical and chemical attributes. Samples were collected at 15 points in each area. Greater activity of urease, acid phosphatase and dehydrogenase were found in the native area. The same result was found for basal respiration, microbial biomass carbon (MBC) and nitrogen (MBN). The structure of Bacteria analyzed by T-RFLP discriminated the native and R20 from R05 and R10, only in the summer. Soil bulk density, humidity and microporosity negatively affected soil microbiological indicators and together with total soil carbon they were the main discriminant factors. A. colossica, A. lacunosa, G. decipiens and Gigaspora sp. were more abundant in NT and the species G. viscosum, A. mellea, A. scrobiculata and S. heterogama in R05 and G. rosea in R10 and R20. Porosity, soil bulk density, humidity, acid phosphatase activity, MBC, MBN and N-NO3 - were the principal environmental variables related to AMF species distribution. Seasonal influences on AMF species were also observed. Higher glomalin related soil protein (GRSP) content was found only in the winter and NT had only EE-GRSP (easly extracted glomalin related soil protein) different from the recovery areas. Correlations among glomalin and physical-chemical and microbiological attributes were observed. Edaphic fauna groups were influenced by seasonality and by sampling methodology (pitfall traps and monoliths). Shannons, Simpsons and the evenness index were significant only in the dry season and in the oldest areas. Richness was higher when the monolith method was used. Diplopoda was the principal group that discriminated the recovery gradient for both seasons and methodologies. Porosity, soil bulk density, humidity, total nitrogen, urease and dehydrogenase were important factors to separate faunal groups. This work showed that biological and physico-chemical soil quality attributes interact and changed according to gradient recovery and seasonality.

Le mutualisme entre les plantes et les champignons arbusculaires mycorhiziens est extrêmement répandu (~ 80% des plantes sont colonisées par ces organismes) et ancien (il ya plus de 450 millions d'années). Cette relation symbiotique est une composante essentielle du fonctionnement des écosystèmes et de leur productivité, et est fortement impliqué dans le cycle de deux éléments clés: le phosphore et le carbone. Le maintien de ce mutualisme est devenu particulièrement important dans le contexte actuel de perte de biodiversité. Un des objectifs de cette thèse était de comprendre la stabilité de ce mutualisme. L'accent a tout d'abord été mis sur les échanges de nutriments impliqués dans cette symbiose, en testant si la plante hôte et les symbiotes fongiques sont capables de discriminer leurs différents partenaires, et d'allouer davantage de ressources aux partenaires fournissant plus de nutriments. J'ai ensuite étudié la possibilité de l'implication de la plante hôte dans la protection des symbiotes mycorhiziens via un transfert de métabolites secondaires dans les hyphes. Nous avons alors pu emettre une nouvelle hypothèse suggérant que la protection en métabolites secondaires venant de la plante serait positivement corrélée avec le niveau de coopération (à savoir le transfert des nutriments) du champignon symbiotique. L'echelle d'étude est ensuite passée de l'individu à la communauté en étudiant les effets de la diminution de la diversité végétale sur la diversité des symbiotes racinaires. Pour ce faire, des analyses moléculaires et des outils novateurs ont été utilisés, tels que le séquençage à haut débit. Pour faciliter encore l'étude des séquences obtenues et d'autres séquences fongiques, j'ai collaboré avec des collègues afin de créer une base de données 'Phymyco-DB' rendue publique en 2012. Enfin, je discute de l'implication du mutualisme mycorhizien dans le contexte des systèmes agricoles actuels et propose de nouvelles trajectoires pour gérer ces systèmes. Ce projet de thèse apporte un nouvel éclairage sur la façon dont fonctionnent ces interactions entre les plantes et champignons MA et sur la manière dont ils façonnent les processus écologiques et les trajectoires évolutives dans les écosystèmes naturels et agricoles. Ces points sont d'une importance majeure pour développer une agriculture plus écologiquement intensive et durable. Le projet a fourni de nouvelles connaissances et perspectives sur la perte de la diversité végétale, et ses conséquences pour la stabilité de la symbiose AM. Comme les champignons mycorhiziens sont essentiels dans les processus des écosystèmes et l'entretien de la fertilité des sols, ce travail devrait avoir un large impact dans (i) la politique de protection des sols, (ii) la recherche sur l'amélioration des plantes et (iii) la conception de systèmes agricoles durables. / The mutualism between plants and arbuscular mycorrhizal fungi is extremely widespread (~ 80% of plants are colonized by these organisms) and ancient (over 450 million years ago). This symbiotic relationship is an essential component of healthy ecosystem functioning and productivity, and is strongly involved in the cycle of two key elements: phosphorus and carbon. Maintaining this mutualism has become especially important in the current context of a biodiversity loss. One goal of this thesis was to understand the stability of the mutualism. I first focused on nutrient exchange, testing whether plant host and fungal symbionts are able to discriminate among partners, and allocate more resources to those individuals providing more nutrients. I then explored the possibility of the host-plant involvement in the protection of mycorrhizal symbionts via a transfer of secondary metabolites into fungal hyphae. We introduced a new hypothesis suggesting that chemcial protection from the plant is positively correlated with the level of cooperation (i.e. nutrient transfer) of the fungal symbiont. I then moved from the individual to the community by studying the effects of decreasing plant diversity on the diversity of root symbionts. To this aim, I utilized molecular analyzes and innovative tools, such as high throughput sequencing. To further facilitate the study of the obtained sequences and other fungal sequences, I worked with colleagues to create a database ‘Phymyco-DB’ which was released to the public in 2012. Finally, I discuss the implication of the mycorrhizal mutualism in the context of current agricultural systems and propose new trajectories to manage these systems. This PhD project provides new insights on how plant and AM fungi interactions work and how they shape ecological processes and evolutionary trajectories in natural and agricultural ecosystems. These points are of major importance to develop a more ecologically intensive agriculture. The project has provided new knowledge and perspectives on the loss of plant diversity, and its consequences for AM symbiosis stability. As arbuscular mycorrhizal fungi are essential in ecosystem processes and soil fertility maintenance, this work should have a broad impact in (i) the soil protection policy, (ii) the research on plant breeding and (iii) the design of sustainable agricultural systems.

Impactos ambientais podem interferir nas características da biomassa microbiana, no processo de ciclagem de nutrientes, nas características físicoquímicas e também na diversidade da microbiota e da macrofauna. O objetivo desse trabalho foi conhecer as diferentes interações entre estes atributos e identificar os indicadores da qualidade do solo envolvidos com o tempo de recuperação. Três áreas com estágios diferentes de recuperação (5, 10 e 20 anos) foram comparadas com uma floresta Estacional Semidecídua nativa (NT) com intuito de estudar o comportamento da microbiota, da macrofauna e de suas interações com os atributos físico-químicos. Foram coletadas amostras em 15 pontos por áreas, escolhidos aleatoriamente. Dentre os atributos microbiológicos, a maior atividade das enzimas urease, fosfatase ácida e desidrogeanse foi encontrada na área nativa. O mesmo foi constatado para a respiração basal e para o carbono e nitrogênio da biomassa microbiana. A análise da estrutura da comunidade de Bacteria, feita a partir de TRFLP, separou as áreas, nativa e de 20 anos de recuperação, das demais, somente no verão. A densidade do solo, a umidade e a microporosidade afetaram negativamente os indicadores microbiológicos do solo, sendo em conjunto com o carbono total do solo os principais fatores discriminantes das áreas. Ocorreu maior presença das espécies de FMA A. spinosa, A. colossica, A. lacunosa, G. decipiens e Gigaspora sp. na área NT e das espécies G. viscosum, A. mellea, A. scrobiculata e S. heterogama na área R05 e G. rosea nas áreas R10 e R20. As principais variáveis ambientais que explicam a relação com as espécies de FMA foram microporosidade, macroporosidade, atividade da fosfatase ácida e densidade, umidade, CBM, NBM e N-NO3. Efeito sazonal sobre as espécies de FMA também foi observado. Maiores valores de proteína do solo relacionada com glomalina (GRSP) foram encontrados no inverno e somente a proteína do solo relacionada com glomalina facilmente extraível (EE-GRSP) separou a área NT das demais. Observou-se, também, alta correlação dessa glicoproteina com os atributos físico-químicos e microbiológicos. Em relação à fauna edáfica ocorreu efeito da sazonalidade e do método de coleta utilizado (armadilhas e monolito). Houve diferença entre os índices de Shanon, Simpson e Pielou somente na época seca, sendo as maiores diferenças nas áreas mais antigas (NT e R20). Maior riqueza foi encontrada ao se utilizar o método de monolito. De acordo com a análise discriminante, Diplopoda foi o principal grupo para as duas épocas e os dois métodos de coleta. Porosidade, densidade basal, umidade, nitrogênio total do solo e atividade das enzimas desidrogenase e urease foram fatores importantes para a separação dos grupos da fauna no gradiente de recuperação ambiental. Esse trabalho mostrou que os atributos biológicos e físicoquímicos de qualidade do solo interagem e se modificam de acordo com a idade das áreas e com a sazonalidade. / Environmental impactation can affect microbial biomass, nutrient cycling, processes, physical-chemical characteristics and also the diversity of microbes and edaphic fauna. The aim of this study was to understand the different interactions between these attributes and to identify the indicators of soil quality involved in the recovery process. Three areas with different stages of recovery [5 (R05), 10 (R10) and 20 (R20) years] were compared with a native semideciduous forest (NT) in order to study the behavior of microbes, macrofauna and their interactions with the physical and chemical attributes. Samples were collected at 15 points in each area. Greater activity of urease, acid phosphatase and dehydrogenase were found in the native area. The same result was found for basal respiration, microbial biomass carbon (MBC) and nitrogen (MBN). The structure of Bacteria analyzed by T-RFLP discriminated the native and R20 from R05 and R10, only in the summer. Soil bulk density, humidity and microporosity negatively affected soil microbiological indicators and together with total soil carbon they were the main discriminant factors. A. colossica, A. lacunosa, G. decipiens and Gigaspora sp. were more abundant in NT and the species G. viscosum, A. mellea, A. scrobiculata and S. heterogama in R05 and G. rosea in R10 and R20. Porosity, soil bulk density, humidity, acid phosphatase activity, MBC, MBN and N-NO3 - were the principal environmental variables related to AMF species distribution. Seasonal influences on AMF species were also observed. Higher glomalin related soil protein (GRSP) content was found only in the winter and NT had only EE-GRSP (easly extracted glomalin related soil protein) different from the recovery areas. Correlations among glomalin and physical-chemical and microbiological attributes were observed. Edaphic fauna groups were influenced by seasonality and by sampling methodology (pitfall traps and monoliths). Shannons, Simpsons and the evenness index were significant only in the dry season and in the oldest areas. Richness was higher when the monolith method was used. Diplopoda was the principal group that discriminated the recovery gradient for both seasons and methodologies. Porosity, soil bulk density, humidity, total nitrogen, urease and dehydrogenase were important factors to separate faunal groups. This work showed that biological and physico-chemical soil quality attributes interact and changed according to gradient recovery and seasonality.

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés. / Spore germination is an essential developmental stage in the life cycle of many filamentous fungi. Arbuscular mycorrhizal fungi (AMF) form a number of different infectious propagules that increase their potential to colonize roots. Among them are extraradical and intraradical spores. The spore cell wall plays a major role in the survival of these propagules by being a physical and osmotic barrier. Because a cell can make considerable adjustments to the composition and structure of its wall in response to environmental conditions, it is possible that intraradical and extraradical spore walls show different mechanical and osmotic properties affecting their survival and germination. However, in contrast to the knowledge on the genetics and molecular composition of AMF cell wall, little is known about its mechanical properties. Information on the germination of AMF under hypertonic conditions is scarce, and experimental designs and methodologies have generally not allowed the direct effects of high external osmotic pressure on fungal germination to be separated from plant-mediated effects. This study had the goal to address two important sets of questions regarding the behavior of mycorrhizal spores. We first determined the relationship between cell wall composition, structure and mechanical properties of the model fungus Glomus irregulare. Micro-indentation was used to quantitatively measure the cell wall mechanical properties. Cell wall composition (chitin and glomalin content) was studied by immunofluorescence whereas optical microscopy was used to measure the cell wall thickness. Glomalin local density and wall thickness were both significantly higher for extraradical spore walls while chitin local density and rigidity were unaffected by origin of spores. High variability in results prevented us from identifying a primary factor responsible for overall wall strength during compression. Decreases of chitin and glomalin concentrations were correlated to the development of the fungal wall throughout its life-cycle. There was also differential association within the wall layers: The chitin and glomalin polymers were localized mostly in the outer and inner walls, respectively. In the second part of our work, we explored the direct effects of fertilizers, in relation to their water activity (aw), on spore germination and cellular turgor pressure. Spores were exposed to three fertilizers with different aw and spore germination and cytorrhysis of spores were assessed after different times of incubation. Water activities of the fertilizers were used as indicators of their osmotic pressures. Osmotic shock exposure of the Glomus irregulare spores to fertilizers at aw values between 0.982 and 0.882 caused gradual changes in cytorrhysis and germination. With the increase of external turgor pressure, cytorrhysis increased while the rate of germination decreased. These effects were most pronounced at high nutrient concentrations. The present investigation, while likely representing a significant step forward in understanding the mechanical and osmotic properties of AMF spores, also confirms that they might depend on many, as yet unidentified factors. Future research should examine differences in the physiology to discern reasons for such differences in spore properties.

Les champignons mycorhiziens à arbuscules (CMA) ont une structure génétique très particulière et certains aspects de leur génétique sont encore incompris et peu documentés. Les CMA se reproduisent par voie asexuée à l’aide de spores multinucléées. Dans cette étude, j’ai cherché à comprendre les mécanismes de l’hérédité génétique des noyaux par la voie de la reproduction asexuée chez les CMA. La première étape était de déterminer le contenu en noyaux des spores matures, ainsi que celui des spores en formation. Des analyses statistiques ont été utilisées pour vérifier le type de relation entre le nombre de noyaux et le diamètre des spores. Quatre espèces du genre Glomus ont été observées au microscope confocal. Les résultats démontrent une hétérogénéité entre les spores dans leur contenu en noyau pour un même diamètre en plus d’une relation positive entre le nombre de noyau et le diamètre de la spore. Afin de vérifier le contenu en noyaux dans les phases extraracinaires, trois différentes structures du mycélium ont été observées au microscope confocal. Aucune structure n’a été retrouvée avec un seul noyau, ce qui permet de conclure que les CMA ne possèdent vraisemblablement pas de stade uninucléé dans leurs phases extraracinaires. Pour étudier l’hérédité des noyaux, deux différentes approches ont été utilisées: (i) Glomus irregulare a été mis sur milieu complémenté avec de l’aphidicoline pour inhiber la mitose. Des observations au microscope confocal ont permis de dénombrer les noyaux qui sont issus des hyphes et non des mitoses. Les résultats indiquent que la population de noyaux présents dans les spores matures provient d’une migration massive de noyaux à l’intérieur des spores en formation suivie d’un nombre faible de mitoses. (ii) La deuxième approche est l’observation microscopique en temps-réel de spores en formation de G. diaphanum qui a permis de confirmer cette affirmation, car il a été possible de voir plusieurs noyaux entrer dans la spore. Dans la dernière partie de cette étude, je me suis intéressée aux échanges génétiques horizontaux chez les CMA qui sont possibles grâce aux anastomoses. Quatre isolats de l’espèce G. irregulare ont été croisés en co-culture par couple de deux isolats (six croisements) pour permettre une proximité propice aux anastomoses et aux échanges génétiques. Ces croisements ont été maintenus pendant deux ans en culture par le repicage des racines colonisées. Des spores des deux différents isolats ont été confrontées sur eau gélifiée, afin d’observer la formation d’anastomose. Un pourcentage de 13% de formation de fusions d’hyphes pour une des confrontations suggère que l’échange des marqueurs parentaux a pu avoir lieu entre les deux isolats grâce aux anastomoses. Un marqueur moléculaire mitochondrial nommé Indel 5 a été développé et utilisé pour l’analyse des spores filles. Ce marqueur possède entre les isolats une délétion de 39 pb et la différence entre les isolats est facilement détectable sur gel d’électrophorèse après amplification PCR. Le génotypage par PCR des spores individuelles a montré que certaines des spores filles issues du croisement possèdent un des deux marqueurs parentaux alors que d’autres spores ont un génotype qui semble posséder les deux marqueurs. Même si la fusion d’hyphes entre spores en germination est possible, d’autres recherches devront être réalisées pour confirmer qu’un échange génétique est possible entre deux isolats très éloignés géographiquement. Le fait qu’il n’existe aucun stade uninucléé au cycle de vie des CMA et qu’il y ait une migration massive de noyaux lors de la formation des spores permet de limiter la dérive génique lors de la reproduction asexuée. Les anastomoses, quant à elles, permettent de rétablir la diversité génétique. Ces deux particularités de la génétique des CMA ont été fort importantes au cours de leur évolution pour permettre de maintenir une variabilité génétique élevée et permettre ainsi une grande adaptation à différents type d’habitats. / Arbuscular Mycorrhizal Fungi (AMF) have a particular and complex genetic structure. Yet, many aspects of their genetics are still misunderstood and poorly documented. These organisms reproduce by asexual multinucleate spores. In this study, I investigated the mechanisms of genetic inheritance of nuclei through asexual reproduction in AMF. First, I determined the number of nuclei in mature and juveniles spores; I used statistical analysis to determine the relationship between the number of nuclei and the spore diameter. Four species from the genus Glomus were observed with a confocal microscope. The results showed that the number of nuclei has a significant positive relationship with spore diameter and more importantly, surprising heterogeneity in the number of nuclei among sister spores was found. To determine the number of nuclei in extraradical phases, three different structures from the mycelia were carefully examined with a confocal microscope. All the structures possessed more than one nucleus and showed that AMF probably lack a single-nucleus stage during their extraradical phases. To study the nuclei’s heritance, two different approaches were used: (i) Glomus irregulare was grown on medium complemented with aphidicolin to inhibit the mitosis. Observations with a confocal microscope permit to count the nuclei that come from the hyphae and not from the mitosis. The results showed that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. (ii) The second approach confirm these results because with time-laps live cellular imaging of young spores of Glomus diaphanum it was possible to see many nuclei to get in the spores. In a second part of this thesis, I studied horizontal gene exchanges among AMF isolates through anastomoses. Thus, four isolates of the species G. irregulare were used in in vitro crossing experiments, in total six combinations using two isolates per crossing experiment. These crossing co-cultures were maintained over two years by subculturing. Spores of two different isolates were confronted in vitro prior to observation of anastomoses. 13% of spores formed anastomoses suggesting the occurrence of genetic exchange between two isolates. A mitochondrial molecular marker referred as Indel 5, was used to genotype individual spores of crossing progenies. A 39 bp deletion occurs in the marker among different isolates and is clearly discriminated by PCR. PCR patterns showed that some spores seem to have both parental markers demonstrating that genetic exchange could occur between the two isolates used in crossing experiment. Even though hyphal fusions occur between germinating spores, subsequent research needs to be done to confirm genetic exchange among different isolates from different geographic areas. The finding that AMF lack a single nuclear stage in their extraradical phases and that mitosis and nuclear migration are the mechanisms by which AMF spores are formed reduce genetic drift that acts on these organisms during asexual reproduction. Anastomoses are likely a mechanism that maintains the genetic diversity in AMF. These genetic characteristics of AMF were very important during their evolution to maintain a high genetic variability that allows their adaptation to many different ecosystems. / 3 vidéos sont dans des fichiers complémentaires à ce mémoire

Made available in DSpace on 2015-03-26T13:36:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 623531 bytes, checksum: 3e10a939648d01ae404b0a09438fda4c (MD5) Previous issue date: 2007-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The high acidity, the intense intemperism, and high aluminum content are limiting factors to plant development in Brazilian Cerrado Upon abiotic stress conditions the association with mycorrhizal fungi can assist the host plant in absorbing nutrients and water from soil. The high availability of these elements to the host plant improved by arbusculares mycorrhizal fungi (AMF) is determinant in plant survival and structuring of plant ecosystems. The presence of dark septate entophytic fungi in plant roots is another type of symbiotic association related to abiotic conditions such as low humid and day length. The reduced number of publications about these associationsin cerrado species is mainly a function of edaphic conditions and root structural characteristics of native species which difficult the in loco studies of these characters. In this work, it was aimed to study the association between AMF and cerrado native species. It was evaluated and adapted methods for fungal structures observation in symbiosis with Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae and Rubiaceae families, which were collected during dry season on senso strictu Cerrado in Floresta Nacional (FLONA) in the municipal district of Paraopeba, Minas Gerais State. Root samples were submitted to three protocols for fungal structures observationg. All evaluated species were colonized by AMF and dark septate fungi, except Xylopia aromatica, which not presented dark septate strutctures. The best diaphanization procedure was obtained when the root samples were autoclaved at 121ºC by 20 minutes in a 2% KOH solution and transferred to a 2% KOH solution by 24 hours at room temperature. This procedure was repeated twice and, after that, the samples were submerged in a 2% KOH solution for 2 hours. The arbuscules were observed with more detail after been included in resin, sectioned and stained with toluidine blue. The non-specific characteristic of AMF observed in sensu stricto Cerrado species was confirmed and suggested for dark septate associations. It indicates that these associations were important as adaptive strategies to cerrado conditions. Roupala montana Aubl. is a species from Proteaceae family which presents a large distribution on different phytophysiognomies of cerrado. Recent studies have been showed that several mycorrhizal fungi structures occurred in these species said to be typically non- mycorrhizal. The low pH and nutrients deficiency in cerrado soils propitiate good conditions for development and establishment of mycorrhizal association in R. montana, as observed in other Proteaceae species. Therefore, in this work it was also aimed to confirm the presence of AMF in R. montana specimens growing at natural cerrado conditions, and correlated the mycorrhizal frequency with nutritional status and edaphic conditions. Three individuals of R. montana were collected in FLONA on cerrado senso strictu, dense cerrado senso strictu, and cerradão during dry and rain seasons. The mycorrhizal occurrence was positively correlated with P, Al, and Mg contents in the soil, and P, K, and Ca in the leaves, and negatively correlated with Ca, Mn, and N contents in the soil. The cerrado soils are acid and have high Al contents which can inhibit the transport of Ca and K to the plants. In this way, the presence of mycorrhizal fungi in R. montana could be a adaptive mechanism for plant surviving under Brazilian cerrado conditions. / A elevada acidez, o intenso intemperismo e os altos teores de alumínio nos solos do Cerrado são fatores limitantes do desenvolvimento vegetal. Em condições de estresses abióticos a associação com fungos micorrízicos auxilia a planta hospedeira na absorção de nutrientes e água do solo. A maior disponibilidades desses elementos para a planta hospedeira pelos fungos micorrízicos arbusculares (FMAs) é determinante na sobrevivência das plantas e na estruturação de formações vegetais. A presença de fungo endofítico do tipo dark septate (DSEF) nas raízes das plantas é outro tipo de associação simbiótica relacionada às condições abióticas como baixa umidade e comprimento do dia. O reduzido número de trabalhos encontrados na literatura dessas associações em espécies de cerrado se deve em parte, pelas condições edáficas e características estruturais radiculares das espécies nativas que dificultam a realização de estudos in loco. Neste trabalho teve-se por objetivo estudar a associação de FMAs em espécies de plantas nativas do cerrado. Para isso, foram testados e adaptados métodos para observação das estruturas fúngicas em simbiose em espécies das famílias Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae e Rubiaceae coletadas durante a estação seca em cerrado senso strictu na Floresta Nacional (FLONA) do município de Paraopeba, Minas Gerais. Amostras radiculares foram submetidas a três tipos de protocolos para observação de estruturas fúngicas. Todas as espécies investigadas encontravam-se colonizadas por FMAs e DSEF, exceto DSEF em Xylopia aromática. O melhor método de diafanização foi observado quando as raízes foram autoclavadas a 121 °C em KOH 2 %, por 20 min e, subseqüentemente transferidas para solução nova de KOH 2 % por 24 horas à temperatura ambiente. Este procedimento foi repetido por duas vezes e, em seguida, essas amostras foram imersas em H2O2 2 % por 2 horas. Os arbúsculos foram observados com maiores detalhes após as raízes serem incluídas em resina, seccionadas e coradas com azul de toluidina. O caráter generalista dos FMAs observado nas espécies vegetais do cerrado sensu stricto foi confirmado e sugerido para DSEFs, indicando a importância destas simbioses como estratégia adaptativa às condições de cerrado. Roupala montana Aubl. é uma espécie da família Proteaceae com ampla distribuição nas diferentes fitofisionomias do cerrado. Estudos recentes têm relatado estruturas típicas de fungos micorrízicos em condições de estresses abióticos em espécies dessa família tipicamente não micorrizável. A acidez e deficiência dos solos de cerrado provem apropriadas condições para o desenvolvimento e formação de associações micorrízicas em R. montana, como observado em outros espécies da família Proteaceae. Assim, este trabalho também teve por objetivo confirmar a presença de fungos micorrízicos arbusculares em R. montana crescidas naturalmente em cerrado, correlacionando a freqüência de micorrização com o estado nutricional e condições edáficas. Em três repetições, indivíduos de R. montana foram coletados na FLONA em cerrado senso strictu, cerrado senso strictu denso e cerradão nas estações seca e chuvosa. A micorrização em R. montana foi correlacionada positivamente com o teor de P, Al, e Mg, no solo e de P, K e Ca nas folhas, e negativamente com Ca, Mn e N no solo. Os solos de Cerrado são ácidos com altas concentrações de Al, os quais podem inibir o transporte de Ca e K para as plantas, sugerindo que a presença de fungos micorrízicos em R. montana é um mecanismo adaptativo de sobrevivência as condições do cerrado brasileiro.

As atividades de mineração causam impactos significativos ao meio ambiente, pois o desenvolvimento dessa atividade implica supressão de vegetação, exposição do solo aos processos erosivos com alterações na quantidade e qualidade dos recursos hídricos superficiais e subterrâneos, além de causar poluição do ar, entre outros aspectos negativos. Uma alternativa para a recuperação de áreas degradadas é a rochagem, que consiste basicamente na incorporação de rochas/minerais ao solo. Uma fonte considerável de resíduos minerais para a rochagem é a indústria de beneficiamento de rochas ornamentais, pois em serrarias que produzem blocos de 20 mm de espessura, de 25 a 30% do bloco de granito são perdidos como partículas finas, considerando o fato de que o mercado brasileiro gera 240.000 t.ano-1, contabilizando apenas o mármore e o granito. O presente trabalho propõe a utilização de resíduos de corte de granito e resíduos orgânicos de Eichhornia crassipes, em consórcio na produção de um substrato rico em matéria orgânica e mineral, composto por micro e macro nutrientes essenciais à nutrição vegetal, favorecendo a recuperação de áreas degradadas por mineração. Desta forma, o presente trabalho objetivou verificar os efeitos da rochagem com resíduos de marmoraria (granito) e da aplicação de resíduos orgânicos de macrófita aquática aguapé (Eichhornia crassipes) sobre aspectos químicos e microbiológicos de um subsolo exposto e sobre o crescimento de capim pensacola (Paspalum notatum), uma gramínea nativa da América do Sul. O experimento foi conduzido em casa de vegetação e constou de 12 tratamentos, como segue: subsolo de área degradada com três doses de resíduos de marmoraria (8, 16 e 32 t. ha-1), combinadas com 8 t. ha-1 de resíduos orgânicos (aguapé), gerando 9 tratamentos, além de três adicionais, com solo de área degradada por mineração de argila vermelha preservado, utilizado como referência. Foram realizadas análises químicas e microbiológicas do solo, além de medidas de altura, massa seca e fresca da parte aérea e do sistema radicular das plantas. Os resultados indicam que os procedimentos com resíduos de granito e aguapé, contribuíram para melhorar as condições químicas e biológicas do solo e proporcionar bons resultados para as variáveis microbiológicas, enquanto as plantas apresentaram o maior crescimento nos tratamentos em que foram adicionados maiores concentrações de resíduos de marmoraria. A rochagem consorciada com macrófitas aquáticas proporcionou elevação da qualidade do solo exposto pela atividade, colaborando para recuperação de locais degradados e fechamento do ciclo de resíduos. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-04-20T13:37:26Z No. of bitstreams: 1 Dissertacao Rogerio Pires Santos.pdf: 3332728 bytes, checksum: 2b1a73f27e95f5e857ab6908bb71178e (MD5) / Made available in DSpace on 2017-04-20T13:37:26Z (GMT). No. of bitstreams: 1 Dissertacao Rogerio Pires Santos.pdf: 3332728 bytes, checksum: 2b1a73f27e95f5e857ab6908bb71178e (MD5) Previous issue date: 2017-04-19 / Mining activities cause significant impacts to the environment, since the development of this activity implies suppression of vegetation, soil exposure to erosive processes with changes in the quantity and quality of surface and underground water resources, besides causing air pollution, among other aspects Negative results. An alternative for the recovery of degraded areas is rock, which basically consists of incorporating rocks / minerals into the soil. A considerable source of mineral waste for rock is the ornamental stone industry, because in sawmills that produce 20 mm thick blocks, 25 to 30% of the granite block is lost as fine particles, considering the fact that The Brazilian market generates 240,000 t.-1, accounting for only marble and granite. The present work proposes the use of granite cutting residues and organic residues of Eichhornia crassipes, in a consortium in the production of a substrate rich in organic and mineral matter, composed of micro and macro nutrients essential to plant nutrition, favoring the recovery of degraded areas by mining. In this way, the present work aimed to verify the effects of rocks with marble and granite residues and the application of organic residues of aquatic macrophyte (Eichhornia crassipes) on chemical and microbiological aspects of an exposed basement and on the growth of pensacola grass (Paspalum notatum), a grass native to South America. The experiment was conducted in a greenhouse and consisted of 12 treatments, as follows: degraded area subsoil with three doses of marble waste (8, 16 and 32. ha- 1), combined with 8 t. ha-1 of organic waste (water hyacinth), generating 9 treatments, in addition to three additional ones, with degraded area soil by mining of preserved red clay, used as reference. Soil chemical and microbiological analyzes were carried out, as well as measurements of height, dry and fresh mass of shoot and root system of plants. The results indicate that the procedures with granite and water hyacinth residues contributed to improve the chemical and biological conditions of the soil and to provide good results for the microbiological variables, while the plants presented the highest growth in the treatments in which were added higher concentrations of residues of marble work. The rock consortium with aquatic macrophytes gave rise to the quality of the soil exposed by the activity, collaborating for the recovery of degraded sites and closure of the waste cycle.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Many studies have shown that inoculation with arbuscular mycorrhizal fungi improved plant growth under salt stress. Considering that the salinity is a serious problem that directly affects the productivity of plants, the aim of this study was to evaluate the effects of inoculation of AMF in maize plants under salt stress in the presence or absence of phosphorus. The experiment was run in a greenhouse of the Department of Biochemist and Molecular Biology of the Federal University of the Cearà (Campus of the Pici, Fortaleza - CearÃ), with four replicates per treatment. The experimental design was completely randomized in factorial arrangement 2 (inoculated and not inoculated plants) x 3 (levels of salinity 0.5, 4.0 and 8.0 dS m-1) x 2 (presence or absence of phosphorus), total of 48 experimental units. During the experiment measurements were made of net photosynthesis, transpiration rate, stomatal conductance and SPAD index. At 40 days after sowing the plants, they were collected, and determined leaf area, shoot dry matter (after drying the material under glass), the relative water content, the osmotic potential, microbiological variables (dependency and mycorrhizal colonization), mineral levels (N, P, K+, Ca2+, Mg2+, Na+, and Cl-) and organic solutes (soluble carbohydrates, N-aminosolubles, proline and soluble protein). The mycorrhizal fungi did not provide an increase in the growth of corn plants, but elevated levels of salinity reduced leaf area and shoot dry matter of plants. Generally, the salinity reduced the photosynthesis, stomatal conductance and transpiration in all treatments. The relative water content was not influenced by any of the factors studied. The SPAD index and relative water content were not influenced by any of the factors studied. The osmotic potential was significantly reduced in treatments with 4.0 and 8.0 dS m-1 salinity compared with the level of 0.5 dS m-1. Mycorrhizal colonization decreased with increasing levels of saline. The levels of glomalin were not influenced by the presence of P and not by increasing levels of salinity. The mycorrhizal fungi did not cause increases in levels of inorganic solutes. The presence of P promoted maize cultivation in non-inoculated plants, increased levels of phosphorus in the leaves and stems, at all levels of salinity. Increased salinity levels decreased the levels of N and Mg2+, but promoted increased levels of Na+ and Cl- in corn plants. The water-soluble carbohydrate showed no significant differences for any of the factors. In general, the levels of N-aminosolubles and proline increased with increasing salinity. Since the levels of soluble proteins showed different responses according to the factors. These results suggest that the mycorrhizal fungi did not minimize the effects of salinity in maize plants (hybrid AG 1051), at least under the conditions employed here. / Muitos estudos tÃm demonstrado que a inoculaÃÃo de plantas com fungos micorrÃzicos arbusculares (FMA) melhora o crescimento das plantas sob estresse salino. Tendo em vista que a salinidade à um problema sÃrio e que afeta de forma direta a produtividade das plantas, o objetivo deste trabalho foi avaliar os efeitos da inoculaÃÃo dos FMA em plantas de milho sob estresse salino, na presenÃa ou ausÃncia de fÃsforo. O experimento foi conduzido na casa de vegetaÃÃo do Departamento de BioquÃmica e Biologia Molecular da Universidade Federal do Cearà (Campus do Pici, Fortaleza - CearÃ), com quatro repetiÃÃes. O delineamento experimental foi o inteiramente casualizado, em arranjo fatorial 2 (plantas inoculadas e nÃo inoculadas) x 3 (nÃveis de salinidade 0,5; 4,0 e 8,0 dS m-1) x 2 (presenÃa ou ausÃncia de fÃsforo), totalizando 48 unidades experimentais. Durante a conduÃÃo do experimento foram realizadas mediÃÃes da fotossÃntese lÃquida, da taxa de transpiraÃÃo, da condutÃncia estomÃtica e do Ãndice SPAD. Aos 40 dias apÃs a semeadura das plantas, as mesmas foram coletadas, sendo determinados a Ãrea foliar, a matÃria seca da parte aÃrea (apÃs secagem do material em estufa), o teor relativo de Ãgua, o potencial osmÃtico, as variÃveis microbiolÃgicas (dependÃncia e colonizaÃÃo micorrÃzica), os teores de alguns elementos minerais (N, P, K+, Ca2+, Mg2+, Na+ e Cl-) e solutos orgÃnicos (carboidratos solÃveis, N-aminossolÃveis, proteÃna solÃvel e prolina). A associaÃÃo micorrÃzica nÃo proporcionou incremento no crescimento das plantas de milho, porÃm os aumentos nos nÃveis de salinidade reduziram a Ãrea foliar e a matÃria seca da parte aÃrea das plantas. De maneira geral, a salinidade reduziu a fotossÃntese, a condutÃncia estomÃtica e a transpiraÃÃo em todos os tratamentos. O Ãndice SPAD e o teor relativo de Ãgua nÃo foram influenciados por nenhum dos fatores estudados. O potencial osmÃtico foi significativamente reduzido nos tratamentos com 4,0 e 8,0 dS m-1 de CE em comparaÃÃo com o nÃvel de 0,5 dS m-1. A colonizaÃÃo micorrÃzica decresceu com o incremento dos nÃveis salinos. Os teores de glomalina nÃo foram influenciados pela presenÃa de P e nem pelos nÃveis crescentes de salinidade. A associaÃÃo micorrÃzica nÃo acarretou incrementos nos teores de solutos inorgÃnicos. A presenÃa de P no cultivo do milho promoveu, nas plantas nÃo inoculadas, aumento nos teores de fÃsforo nas folhas e nos colmos, em todos os nÃveis de salinidade. O aumento dos nÃveis de salinidade reduziu os teores de N e Mg2+, porÃm promoveram o aumento nos teores de Na+ e Cl- nas plantas de milho. Os teores de carboidratos solÃveis nÃo apresentaram diferenÃas significativas para nenhum dos fatores analisados. De modo geral, os teores de N-aminossolÃveis e de prolina aumentaram com o incremento da salinidade. Jà os teores de proteÃnas solÃveis apresentaram respostas diferenciadas de acordo com os fatores analisados. Esses resultados sugerem que a associaÃÃo micorrÃzica nÃo minimizou os efeitos da salinidade nas plantas de milho (hÃbrido AG 1051), pelo menos nas condiÃÃes aqui empregadas.

As atividades de mineração causam impactos significativos ao meio ambiente, pois o desenvolvimento dessa atividade implica supressão de vegetação, exposição do solo aos processos erosivos com alterações na quantidade e qualidade dos recursos hídricos superficiais e subterrâneos, além de causar poluição do ar, entre outros aspectos negativos. Uma alternativa para a recuperação de áreas degradadas é a rochagem, que consiste basicamente na incorporação de rochas/minerais ao solo. Uma fonte considerável de resíduos minerais para a rochagem é a indústria de beneficiamento de rochas ornamentais, pois em serrarias que produzem blocos de 20 mm de espessura, de 25 a 30% do bloco de granito são perdidos como partículas finas, considerando o fato de que o mercado brasileiro gera 240.000 t.ano-1, contabilizando apenas o mármore e o granito. O presente trabalho propõe a utilização de resíduos de corte de granito e resíduos orgânicos de Eichhornia crassipes, em consórcio na produção de um substrato rico em matéria orgânica e mineral, composto por micro e macro nutrientes essenciais à nutrição vegetal, favorecendo a recuperação de áreas degradadas por mineração. Desta forma, o presente trabalho objetivou verificar os efeitos da rochagem com resíduos de marmoraria (granito) e da aplicação de resíduos orgânicos de macrófita aquática aguapé (Eichhornia crassipes) sobre aspectos químicos e microbiológicos de um subsolo exposto e sobre o crescimento de capim pensacola (Paspalum notatum), uma gramínea nativa da América do Sul. O experimento foi conduzido em casa de vegetação e constou de 12 tratamentos, como segue: subsolo de área degradada com três doses de resíduos de marmoraria (8, 16 e 32 t. ha-1), combinadas com 8 t. ha-1 de resíduos orgânicos (aguapé), gerando 9 tratamentos, além de três adicionais, com solo de área degradada por mineração de argila vermelha preservado, utilizado como referência. Foram realizadas análises químicas e microbiológicas do solo, além de medidas de altura, massa seca e fresca da parte aérea e do sistema radicular das plantas. Os resultados indicam que os procedimentos com resíduos de granito e aguapé, contribuíram para melhorar as condições químicas e biológicas do solo e proporcionar bons resultados para as variáveis microbiológicas, enquanto as plantas apresentaram o maior crescimento nos tratamentos em que foram adicionados maiores concentrações de resíduos de marmoraria. A rochagem consorciada com macrófitas aquáticas proporcionou elevação da qualidade do solo exposto pela atividade, colaborando para recuperação de locais degradados e fechamento do ciclo de resíduos. / Mining activities cause significant impacts to the environment, since the development of this activity implies suppression of vegetation, soil exposure to erosive processes with changes in the quantity and quality of surface and underground water resources, besides causing air pollution, among other aspects Negative results. An alternative for the recovery of degraded areas is rock, which basically consists of incorporating rocks / minerals into the soil. A considerable source of mineral waste for rock is the ornamental stone industry, because in sawmills that produce 20 mm thick blocks, 25 to 30% of the granite block is lost as fine particles, considering the fact that The Brazilian market generates 240,000 t.-1, accounting for only marble and granite. The present work proposes the use of granite cutting residues and organic residues of Eichhornia crassipes, in a consortium in the production of a substrate rich in organic and mineral matter, composed of micro and macro nutrients essential to plant nutrition, favoring the recovery of degraded areas by mining. In this way, the present work aimed to verify the effects of rocks with marble and granite residues and the application of organic residues of aquatic macrophyte (Eichhornia crassipes) on chemical and microbiological aspects of an exposed basement and on the growth of pensacola grass (Paspalum notatum), a grass native to South America. The experiment was conducted in a greenhouse and consisted of 12 treatments, as follows: degraded area subsoil with three doses of marble waste (8, 16 and 32. ha- 1), combined with 8 t. ha-1 of organic waste (water hyacinth), generating 9 treatments, in addition to three additional ones, with degraded area soil by mining of preserved red clay, used as reference. Soil chemical and microbiological analyzes were carried out, as well as measurements of height, dry and fresh mass of shoot and root system of plants. The results indicate that the procedures with granite and water hyacinth residues contributed to improve the chemical and biological conditions of the soil and to provide good results for the microbiological variables, while the plants presented the highest growth in the treatments in which were added higher concentrations of residues of marble work. The rock consortium with aquatic macrophytes gave rise to the quality of the soil exposed by the activity, collaborating for the recovery of degraded sites and closure of the waste cycle.