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 hydrocarbures aromatiques polycyliques (HAPs) figurent parmi les polluants organiques persistants majeurs des sols pollués et présentent une toxicité avérée vis-à-vis de l'homme et des écosystèmes. Parmi les méthodes de remédiation des sols pollués par les HAPs, la phytoremédiation assistée par les champignons mycorhiziens à arbuscules (CMA), pourrait représenter une alternative innovante, écologique et économique. L'utilisation des mycorhizes comme outil de phytoremédiation des sols pollués présente plusieurs avantages dont une meilleure tolérance à la toxicité des HAPs, une meilleure nutrition hydrique et minérale ainsi qu'une meilleure dissipation des HAPs. De rares études ont décrit l'impact des HAPs sur le développement des CMA en lien avec une péroxydation lipidique et une perturbation des teneurs en lipides du CMA, mais ni les cibles d'action de ces polluants au niveau du métabolisme lipidique, ni le rôle de ces modifications dans sa tolérance aux HAPs et dans leur dissipation n'ont été étudiés. C'est pourquoi, le premier objectif de ce travail vise tout d'abord à comprendre l'impact des HAPs sur le métabolisme lipidique. Le radiomarquage par l'acétate [1-¹⁴C] a permis de montrer une perturbation de la biosynthèse des lipides membranaires du CMA extra-racinaire. D'autre part, nos résultats montrent que les HAPs affectent la nutrition phosphatée. Par ailleurs, la capacité des mycorhizes à dégrader et à bioaccumuler le benzo[a]pyrène est démontrée. Enfin, l'implication du métabolisme des lipides de réserve (les triacylglycérols) du mycélium extra-racinaire dans la régénération des membranes altérées, la lutte contre le stress oxydant induit par les HAPs et dans leur métabolisation/bioaccumulation est discutée. / Polycyclic aromatic hydrocarbons (PAHs) are among the major persistent organic pollutant frequently found in the polluted soils and are harmful for human health and its environment. To clean-up the PAHs polluted soils, phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) could represent an innovative, ecological and cost-effective alternative. The use of mycorrhizas, as phytoremediation tool, has several advantages including increased tolerance to the pollutant toxicity, improved water and mineral nutrition as well as a better pollutant dissipation. Few studies have described the impact of PAHs on the AMF development related with lipid peroxidation and total lipid content disturbance. However, so far neither the target action of these pollutants on the metabolism, nor the role of these lipid changes in PAH tolerance and in their dissipation have been studied. Therefore, the present work aims firstly to improve our understanding of the PAHs impact on the CMA lipid metabolism. Thanks to radiolabeling experiments with [1-¹⁴C] acetate, our results showed a disruption of the membrane lipid biosynthesis pathways in the AMF extraradical mycelium, grown in the presence of PAHs. Secondly, it was highlighted that the PAHs affectef the phosphate nutrition. Finally, the mycorrhizas abilities to degrade and to bioaccumulate the benzo[a]pyrene, were pointed out. The involvement of extraradical mycelium storage lipid (triacyglycerols) metabolism in the membrane regeneration, the fight against the PAH induced-oxidative stress and the PAH metabolism/bioaccumulation is discussed.

Les plantes n'exploitent pas seules les nutriments du sol, mais dépendent de champignons avec lesquels elles forment des symbioses mycorhiziennes dans leurs racines. C'est en particulier vrai pour les 25 000 espèces d'orchidées actuelles qui dépendent toutes de champignons mycorhiziens pour accomplir leur cycle de vie. Elles produisent des graines microscopiques qui n'ont pas les ressources nutritives pour germer, mais qui dépendent de la présence de partenaires adéquats pour nourrir l'embryon (hétérotrophie) jusqu'à l'apparition des feuilles (autotrophie). Les mycorhiziens restent présents dans les racines des adultes où ils contribuent à la nutrition, ce qui permet d'étudier plus facilement la diversité des symbiotes à l'aide des outils génétiques. Conscients des biais des études en faveur des régions tempérées, nous avons étudié la diversité des mycorhiziens d'orchidées tropicales à La Réunion. Nous avons montré que (1) les orchidées tropicales ont des partenaires semblables aux orchidées tempérées et méditerranéennes (Sebacinales, Ceratobasidiaceae et surtout Tulasnellaceae), et que ces taxons de champignons sont largement représentés dans différents biomes et dans différentes plantes hôtes. Nous avons aussi démontré pour la première fois que (2) les orchidées épiphytes (dont les associations étaient peu connues) ont des cortèges mycorhiziens différents de ceux des orchidées terrestres dans les communautés tropicales. De plus, en développant une approche à l'échelle de réseaux d'interactions (78 espèces de La Réunion), nous avons montré que (3) les espèces tropicales ont tendance à être généralistes et que (4) le réseau mycorhizien des orchidées montre des propriétés semblables à celles des réseaux d'interactions mutualistes (nestedness et asymétrie d'interaction), alors que la nature mutualiste de cette symbiose mycorhiziennes fait débat. Dans un second volet de la thèse, nous avons étudié les partenaires des orchidées non chlorophylliennes (mycohétérotrophes) tropicales. Nous avons montré que (5) les espèces tropicales peuvent s'associer à des champignons saprophytes qui les nourrissent en carbone issu de la décomposition de la litière dans les forêts tropicales humides et que (6) les modèles tropicaux (en n'étant pas spécifiques) remettent en question les idées reçues sur la mycohétérotrophie des plantes. Nous avons confirmé que (7) la mycohétérotrophie dérive d'un régime nutritionnel intermédiaire (mixotrophie) mis en place dans des lignées chlorophylliennes. Dans un dernier volet de la thèse, nous avons posé la question du déterminisme phylogénétique des associations orchidées-champignons. En analysant la force du signal dans les phylogénies des deux partenaires, nous avons vérifié que (8) les associations mycorhiziennes sont peu conservées à l'échelle supra-générique dans la phylogénie des orchidées, et qu'elles (9) peuvent être maintenues à une échelle plus récente (cas de certains clades d'angraecoïdes). Ces résultats soulignent l'empreinte relative des processus écologiques et évolutifs sur les patrons d'associations actuels, et remettent en question l'idée qu'un processus de coévolution pourrait guider le système. / Plants generally do not exploit soil nutrients themselves, but they depend upon mycorrhizal symbioses with root-associating fungi. This is the case for the current 25,000 orchid species that depend on the development of a mycorrhizal association to germinate and establish. They produce minute seeds lacking nutritional ressources required to germinate, so that they depend on the presence of suitable fungal partners to obtain carbon (heterotrophy) until the development of leaves (autotrophy). Mycorrhizal fungi remain present in the roots of adult plants where they contribute to the plant nutrition, which makes the molecular identification of fungal partners easier. Given the fact that most studies have been conducted in temperate regions, we have studied the diversity of mycorrhizal fungi in tropical orchids of La Réunion. We have found that (i) tropical orchids have the same partners as temperate and mediterranean orchids (Sebacinales, Ceratobasidiaceae, and above all Tulasnellaceae), and that these fungi are widespread in biomes and host plants. We have also showed for the first time that (ii) epiphytic orchids-that have hardly been studied-have partners that differ from terrestrial orchids' in tropical plant communities. Moreover, by developing an interaction network approach (78 species of La Réunion), we have found that (iii) most tropical species are generalists and that (iv) the mycorrhizal network shows the same properties as the mutualistic interaction networks' (nestedness and interaction asymmetry), whereas the mutualistic nature of the orchid symbiosis is still a current issue. In the second part of our thesis, we have studied the fungal partners of achlorophyllous (i.e. mycoheterotrophic) tropical orchids. We have found that (v) tropical species often associate with saprophytic fungi that provide carbon extracted from decaying wood or leaves in tropical soils, and that (vi) tropical models, because of their lack of specificity, challenge the rule drawn from temperate models. We have also confirmed in tropical models that (vii) mycoheterotrophy evolved from mixotrophic ancestors (i.e. intermediate nutritional mode). In the last part of our thesis, we have dealt with the influence of orchid and fungal phylogenies in explaining the structure of the observed networks. By measuring the phylogenetic signals in both orchid and fungal phylogenies, we have checked that (viii) mycorrhizal interactions are not explained by the phylogenetic placements of either orchid genera or fungal taxa. However, we have noticed that (ix) a phylogenetic signal can occur in recent clades of orchid species (but not in fungal species). These results provide insights in the relative imprint of ecological and evolutionary processes on the current patterns of fungus-orchid associations, and challenge the idea that the coevolutionary process could drive the system.

No description available.

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.

Os fungos micorrízicos arbusculares (FMA) são muito importantes para a nutrição mineral das plantas e para a recuperação de áreas degradadas, pois possibilitam um melhor desenvolvimento das espécies arbóreas usadas na revegetação. Entretanto, para um melhor desempenho dos FMA, as condições ambientais devem ser favoráveis, pois tanto a ocorrência quanto a diversidade desses fungos são bastante influenciados por diversos fatores como os químicos, físico e microbiológico do solo. Diferentes profundidades do solo devem ser consideradas para um levantamento mais detalhado. Avaliaram-se os atributos químicos, físicos e microbiológicos e, entre estes, a glomalina e a ocorrência e diversidade de FMA em diferentes profundidades (0-15 cm, 15-30 cm e 30-45 cm). As áreas selecionadas para estudo foram: nativa (NT) e em gradiente de recuperação ambiental: 5 anos (R05), 10 anos (R10) e 20 anos (R20), todas localizadas no Bioma Mata Atlântica, Estado de São Paulo, Brasil. As coletas de solo e raízes foram realizadas em dois períodos: janeiro (chuvoso) e junho (seco). A colonização radicular foi avaliada em três pontos de amostragem sendo: (A) na base do tronco, (B) distanciadas em 1m e (C) de plantas no entorno das árvores. Os esporos foram identificados e posteriormente foram calculados a abundância relativa, e os índices de riqueza (R), diversidade de Shannon (H) e dominância de Simpson (Is) das espécies de FMA. Os dados foram submetidos à análise de variância ANOVA, teste t, análise canônica discriminante (ACD) e análise de redundância (RDA). A colonização radicular foi maior para as áreas em estádios mais avançados de recuperação, nas raízes coletadas no ponto C e na época seca. A densidade de esporos de FMA, na época chuvosa, foi maior em R05 em todas as profundidades, enquanto que na época seca a densidade de esporos foi maior para R05 apenas em 0-15 cm, a partir de 15 cm uma maior densidade de esporos foi verificada para NT e R10. Considerando os dois períodos amostrados e as quatro áreas estudadas, foram identificadas 23 espécies de FMA, sendo os gêneros Glomus e Acaulospora os mais freqüentes. Um maior índice de riqueza (R) e diversidade (H) de espécies foi encontrado na área NT e nem sempre uma maior diversidade foi verificada nas camadas mais superficiais. Espécies do gênero Acaulospora (Ac. mellea e Ac. scrobiculata) e Glomus (G. viscosum e G. sp2.) foram sempre abundantes em todas as profundidades analisadas, enquanto que as espécies do gênero Gigaspora, Ambispora e Racocetra foram raras em todas as profundidades. A ACD indicou que Glomus viscosum, Acaulospora scrobiculata, Acaulospora mellea e Scutellospora heterogama foram as espécies que mais contribuíram para distinguir as áreas e a RDA mostrou que dentre os atributos do solo, o pH, H+Al, carbono do solo, glomalina, umidade, microporosidade e densidade foram os que mais influenciaram a diversidade de FMA. A umidade, densidade e glomalina correlacionaram positivamente com o número de esporos, já o nitrato do solo apresentou uma correlação negativa. / Arbuscular mycorrhizal fungi (AMF) are very important for the recuperation of degraded land, because they enable better absorption of mineral nutrients by plants and favor the development of tree species used in revegetation.However, for greater efficiency of mycorrhizae the environmental conditions must be favorable, because the occurrence and diversity of these fungi are greatly influenced by several factors such as chemical, physical and microbiological soil attributes. Different soil depths should be considered for a more detailed survey. Chemical, physical and microbiological evaluations, including the soil glomalin content and the diversity of AMF at different depths (0-15 cm, 15-30 cm and 30-45 cm) were performed. The areas selected for study were: native forest (NT) and a gradient of environmental restoration and reforestation: five years (R05), 10 years (R10) and 20 (R20), all located in the Atlantic forest, State of Sao Paulo, Brazil. The samples of soil and roots were taken in two periods: January (rainy) and June (dry season). Root colonization was assessed at three sites as follows: (A) at the base of the tree trunk, (B) 1m away from the trunk, and (C) seedlings growing around the adult trees. Spores were identified and the relative abundance and richness indices (R), Shannons diversity (H) and Simpsons dominance index (Is) of AMF were calculated. The data were analyzed using ANOVA, the t test, canonical discriminant analysis (CDA) and redundancy analysis (RDA). The root colonization was higher for areas with more advanced stages of recovery, in roots collected at point C and during the dry season. The density of spores in the rainy season was higher in R05 at all depths, while in the dry season the density of spores was higher for R05 only at 0-15 cm, and from 15 cm downwards a higher density of spores was observed for NT and R10.Considering the two periods and the four areas studied, we identified 23 AMF species, and the genera Glomus and Acaulospora were the most frequent. A higher richness (R) and diversity index (H) of species was found in the area NT. Not always the greatest diversity was found in the superficial layers. Species of Acaulospora (Ac. scrobiculata and Ac. mellea), Glomus (Glomus sp2. and G. viscosum) were always abundant at all depths, while the species of the genera Gigaspora, Ambispora and Racocetra were rare at all depths. The CDA indicated that Glomus viscosum, Acaulospora scrobiculata, Acaulospora mellea and Scutellospora heterogama were the species that contributed most to distinguishing the areas and the RDA showed that among the soil characteristics, pH, H + Al, soil carbon, glomalin, humidity, microporosity and density were the attributes that most influenced the diversity of AMF. Moisture, density and glomalin were positively correlated with the number of spores, however, the soil nitrate showed a negative correlation.

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.

Os fungos micorrízicos arbusculares (FMA) são muito importantes para a nutrição mineral das plantas e para a recuperação de áreas degradadas, pois possibilitam um melhor desenvolvimento das espécies arbóreas usadas na revegetação. Entretanto, para um melhor desempenho dos FMA, as condições ambientais devem ser favoráveis, pois tanto a ocorrência quanto a diversidade desses fungos são bastante influenciados por diversos fatores como os químicos, físico e microbiológico do solo. Diferentes profundidades do solo devem ser consideradas para um levantamento mais detalhado. Avaliaram-se os atributos químicos, físicos e microbiológicos e, entre estes, a glomalina e a ocorrência e diversidade de FMA em diferentes profundidades (0-15 cm, 15-30 cm e 30-45 cm). As áreas selecionadas para estudo foram: nativa (NT) e em gradiente de recuperação ambiental: 5 anos (R05), 10 anos (R10) e 20 anos (R20), todas localizadas no Bioma Mata Atlântica, Estado de São Paulo, Brasil. As coletas de solo e raízes foram realizadas em dois períodos: janeiro (chuvoso) e junho (seco). A colonização radicular foi avaliada em três pontos de amostragem sendo: (A) na base do tronco, (B) distanciadas em 1m e (C) de plantas no entorno das árvores. Os esporos foram identificados e posteriormente foram calculados a abundância relativa, e os índices de riqueza (R), diversidade de Shannon (H) e dominância de Simpson (Is) das espécies de FMA. Os dados foram submetidos à análise de variância ANOVA, teste t, análise canônica discriminante (ACD) e análise de redundância (RDA). A colonização radicular foi maior para as áreas em estádios mais avançados de recuperação, nas raízes coletadas no ponto C e na época seca. A densidade de esporos de FMA, na época chuvosa, foi maior em R05 em todas as profundidades, enquanto que na época seca a densidade de esporos foi maior para R05 apenas em 0-15 cm, a partir de 15 cm uma maior densidade de esporos foi verificada para NT e R10. Considerando os dois períodos amostrados e as quatro áreas estudadas, foram identificadas 23 espécies de FMA, sendo os gêneros Glomus e Acaulospora os mais freqüentes. Um maior índice de riqueza (R) e diversidade (H) de espécies foi encontrado na área NT e nem sempre uma maior diversidade foi verificada nas camadas mais superficiais. Espécies do gênero Acaulospora (Ac. mellea e Ac. scrobiculata) e Glomus (G. viscosum e G. sp2.) foram sempre abundantes em todas as profundidades analisadas, enquanto que as espécies do gênero Gigaspora, Ambispora e Racocetra foram raras em todas as profundidades. A ACD indicou que Glomus viscosum, Acaulospora scrobiculata, Acaulospora mellea e Scutellospora heterogama foram as espécies que mais contribuíram para distinguir as áreas e a RDA mostrou que dentre os atributos do solo, o pH, H+Al, carbono do solo, glomalina, umidade, microporosidade e densidade foram os que mais influenciaram a diversidade de FMA. A umidade, densidade e glomalina correlacionaram positivamente com o número de esporos, já o nitrato do solo apresentou uma correlação negativa. / Arbuscular mycorrhizal fungi (AMF) are very important for the recuperation of degraded land, because they enable better absorption of mineral nutrients by plants and favor the development of tree species used in revegetation.However, for greater efficiency of mycorrhizae the environmental conditions must be favorable, because the occurrence and diversity of these fungi are greatly influenced by several factors such as chemical, physical and microbiological soil attributes. Different soil depths should be considered for a more detailed survey. Chemical, physical and microbiological evaluations, including the soil glomalin content and the diversity of AMF at different depths (0-15 cm, 15-30 cm and 30-45 cm) were performed. The areas selected for study were: native forest (NT) and a gradient of environmental restoration and reforestation: five years (R05), 10 years (R10) and 20 (R20), all located in the Atlantic forest, State of Sao Paulo, Brazil. The samples of soil and roots were taken in two periods: January (rainy) and June (dry season). Root colonization was assessed at three sites as follows: (A) at the base of the tree trunk, (B) 1m away from the trunk, and (C) seedlings growing around the adult trees. Spores were identified and the relative abundance and richness indices (R), Shannons diversity (H) and Simpsons dominance index (Is) of AMF were calculated. The data were analyzed using ANOVA, the t test, canonical discriminant analysis (CDA) and redundancy analysis (RDA). The root colonization was higher for areas with more advanced stages of recovery, in roots collected at point C and during the dry season. The density of spores in the rainy season was higher in R05 at all depths, while in the dry season the density of spores was higher for R05 only at 0-15 cm, and from 15 cm downwards a higher density of spores was observed for NT and R10.Considering the two periods and the four areas studied, we identified 23 AMF species, and the genera Glomus and Acaulospora were the most frequent. A higher richness (R) and diversity index (H) of species was found in the area NT. Not always the greatest diversity was found in the superficial layers. Species of Acaulospora (Ac. scrobiculata and Ac. mellea), Glomus (Glomus sp2. and G. viscosum) were always abundant at all depths, while the species of the genera Gigaspora, Ambispora and Racocetra were rare at all depths. The CDA indicated that Glomus viscosum, Acaulospora scrobiculata, Acaulospora mellea and Scutellospora heterogama were the species that contributed most to distinguishing the areas and the RDA showed that among the soil characteristics, pH, H + Al, soil carbon, glomalin, humidity, microporosity and density were the attributes that most influenced the diversity of AMF. Moisture, density and glomalin were positively correlated with the number of spores, however, the soil nitrate showed a negative correlation.

Made available in DSpace on 2017-07-10T17:37:33Z (GMT). No. of bitstreams: 1 Luciana_Cleci_de_Oliveira.pdf: 753219 bytes, checksum: cc1a5575b73ca9b97e749c6fa03d3cd3 (MD5) Previous issue date: 2009-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due the lack of informatios about fertilization on lettuce (Lactuca sativa L.) in organic growth, this work aimed to study the effect of organic fertilizers on chemical and biological character of soil and on lettuce productivity. The green manure velvet bean (Mucuna aterrima) received two types of management: incorporation into the soil or not incorporation. These both areas received the solid organic fertilizer bovine manure, commercial organic fertilizer and organic compound and a control treatment without fertilization. The samples were obtained at the time of management of velvet bean, and at 15, 55 and 105 days after this process, representing the moments of the distribution of fertilizes, plant and harvest of lettuce, respectively. Were evaluated chemical and biological characteristics of soil, and agronomic characteristics of the lettuce cultivar Vanda. The results showed that the commercial organic fertilizer followed by the organic compound, provided that highest amorents of organic matter, P, K, Ca, Mg, CTC, pH and V, and the two types of management of velvet bean, incorporated or not has similar results. For microbial biomass, better results were fownd with velvet bean not incorporated into soil plus treatment with organic compound. The population of bacteria was increased in the area where velvet bean was incorporated, except for the treatment with organic compound. For fungi, not incorporated velvet bean associated with commercial organic fertilizer provided the higher rate of population. The incorporation of velvet bean associated with organic compound increased the population of mycorrhizal fungi. The bovine manure associated with the incorporation of velvet bean and treatment with organic compound without incorporation of velvet bean increased the population of saprophytic nematodes. The area of aerial part, number of leaves and total fresh and dry weights of aerial part of lettuce, higher values were observed without the incorporation of velvet bean associated with the use of commercial organic fertilizer or bovine manure treatmentes / Em função da carência de informações técnicas sobre adubação da cultura da alface (Lactuca sativa L.), em sistema de cultivo orgânico, este trabalho teve o objetivo de estudar práticas mais convenientes de adubação que auxiliem na manutenção da fertilidade do solo e produtividade da cultura. O delineamento experimental foi o de blocos ao acaso em parcelas subdivididas onde o adubo verde mucuna preta (Mucuna aterrima) recebeu dois tipos de manejo: incorporação ao solo seguida do levantamento de canteiros, e não incorporação. As subparcelas receberam os adubos orgânicos sólidos: esterco bovino, adubo orgânico comercial e composto além da testemunha sem adubação, com cinco repetiçiões, sendo utilizada a alface cultivar Vanda. As épocas de avaliação ocorreram no momento do manejo da mucuna, e aos 15, 55 e 105 dias após esse manejo, representando momentos da distribuição dos adubos, plantio e colheita da alface, respectivamente. Foram avaliadas características químicas e biológicas do solo, e características fitométricas da planta. Pelos resultados observou-se que o adubo orgânico comercial seguido do composto, foram os que proporcionaram índices superiores de matéria orgânica, P, K, Ca, Mg, CTC, V e pH, e o manejo diferenciado do adubo verde, com ou sem incorporação possibilitou resultados semelhantes. Para a biomassa microbiana, resultados superiores foram observados com a mucuna sem incorporação e o tratamento com composto. A população de bactérias foi aumentada para o manejo da mucuna com incorporação, exceto para o tratamento composto. Para fungos, o manejo sem incorporação associado ao adubo orgânico comercial proporcionou maiores índices populacionais. O manejo com incorporação associado ao composto se mostraram favoráveis para aumento da população de fungos micorrízicos. O esterco bovino associado a incorporação da mucuna e o tratamento com composto sem incorporação da mucuna aumentaram a população de nematóides saprófitas. Para a área de projeção de saia, número de folhas e massa fresca e seca totais da parte aérea, foram observados valores superiores sem a incorporação da mucuna preta e para os tratamentos adubo orgânico comercial e esterco bovino