Thesis (PhD (Forest and Wood Science))—University of Stellenbosch, 2006. / Experiments carried out in this study sought to contribute to the understanding of the ecological interactions involved in Uapaca kirkiana seedling establishment and growth in a natural woodland ecosystem. These include soil pH reactions, plant root surface pH change, root exudates that affect the chemical behaviour of the soil in the vicinity of the root, and microbial effects. Although it was difficult to determine which of these factors play a dominant role in the soil-plant relationship, the study contributed to the understanding of the mycorrhizal fungi-host plant association. It revealed the diversity of mycorrhizal fungal species occurring in the different ecological sites, and also analysed the relationship between soil factors. Soil pH and K had significant influences on the fungi population diversity. K was considered important in its role in the translocation of auxins to the root sites where they enhance root susceptibility to mycorrhizal fungal infection. More work needs to be undertaken to establish the role of soil pH.

Les métaux lourds (ML) s’accumulent de plus en plus dans les sols à l’échelle mondiale, d’une part à cause des engrais minéraux et divers produits chimiques utilisés en agriculture intensive, et d’autre part à cause des activités industrielles. Toutes ces activités génèrent des déchets toxiques qui s’accumulent dans l’environnement. Les ML ne sont pas biodégradables et leur accumulation cause donc des problèmes de toxicité des sols et affecte la biodiversité des microorganismes qui y vivent. La fertilisation en azote (N) est une pratique courante en agriculture à grande échelle qui permet d’augmenter la fertilité des sols et la productivité des cultures. Cependant, son utilisation à long terme cause plusieurs effets néfastes pour l'environnement. Par exemple, elle augmente la quantité des ML dans les sols, les nappes phréatiques et les plantes. En outre, ces effets néfastes réduisent et changent considérablement la biodiversité des écosystèmes terrestres. La structure des communautés des champignons mycorhiziens à arbuscules (CMA) a été étudiée dans des sols contaminés par des ML issus de la fertilisation à long terme en N. Le rôle des différentes espèces de CMA dans l'absorption et la séquestration des ML a été aussi investigué. Dans une première expérience, la structure des communautés de CMA a été analysée à partir d’échantillons de sols de sites contaminés par des ML et de sites témoins non-contaminés. Nous avons constaté que la diversité des CMA indigènes a été plus faible dans les sols et les racines des plantes récoltées à partir de sites contaminés par rapport aux sites noncontaminés. Nous avons également constaté que la structure de la communauté d'AMF a été modifiée par la présence des ML dans les sols. Certains ribotypes des CMA ont été plus souvent associés aux sites contaminés, alors que d’autres ribotypes ont été associés aux sites non-contaminés. Cependant, certains ribotypes ont été observés aussi bien dans les sols pollués que non-pollués. Dans une deuxième expérience, les effets de la fertilisation organique et minérale (N) sur les différentes structures des communautés des CMA ont été étudiés. La variation de la structure de la communauté de CMA colonisant les racines a été analysée en fonction du type de fertilisation. Certains ribotypes de CMA étaient associés à la fertilisation organique et d'autres à la fertilisation minérale. En revanche, la fertilisation minérale a réduit le nombre de ribotypes de CMA alors que la fertilisation organique l’a augmenté. Dans cette expérience, j’ai démontré que le changement de structure des communautés de CMA colonisant des racines a eu un effet significatif sur la productivité des plantes. Dans une troisième expérience, le rôle de deux espèces de CMA (Glomus irregulare et G. mosseae) dans l'absorption du cadmium (Cd) par des plants de tournesol cultivés dans des sols amendés avec trois niveaux différents de Cd a été évalué. J’ai démontré que les deux espèces de CMA affectent différemment l’absorption ou la séquestration de ce ML par les plants de tournesol. Cette expérience a permis de mieux comprendre le rôle potentiel des CMA dans l'absorption des ML selon la concentration de cadmium dans le sol et les espèces de CMA. Mes recherches de doctorat démontrent donc que la fertilisation en N affecte la structure des communautés des CMA dans les racines et le sol. Le changement de structure de la communauté de CMA colonisant les racines affecte de manière significative la productivité des plantes. J’ai aussi démontré que, sous nos conditions expériemntales, l’espèce de CMA G. irregulare a été observée dans tous les sites (pollués et non-pollués), tandis que le G. mosseae n’a été observé en abondance que dans les sites contaminés. Par conséquent, j’ai étudié le rôle de ces deux espèces (G. irregulare et G. mosseae) dans l'absorption du Cd par le tournesol cultivé dans des sols amendés avec trois différents niveaux de Cd en serre. Les résultats indiquent que les espèces de CMA ont un potentiel différent pour atténuer la toxicité des ML dans les plantes hôtes, selon le niveau de concentration en Cd. En conclusion, mes travaux suggèrent que le G. irregulare est une espèce potentiellement importante pour la phytoextration du Cd, alors que le G. mosseae pourrait être une espèce appropriée pour phytostabilisation du Cd et du Zn. / Trace metals (TM) are continually world-wide added to soils through the intensive use of mineral fertilizers and agriculture chemicals, together with industrial and other activities generating toxic wastes. Problems associated with metal-contaminated soil exists because TM are not biodegradable. TM that accumulate in soils affect the biodiversity of soil microorganisms. Nitrogen (N) fertilization is a widespread practice to increase soil fertility and crop production. However, the long-term use of N fertilization causes many detrimental effects in the environment. The intensive use of N fertilization increase TM input in soils, and in extreme cases, N fertilization result in TM pollution of the surrounding soil and water and increase TM concentration in plant tissues. In addition, the long-term use of N fertilizers changes and declines the biodiversity of above and underground ecosystems. The community structure of arbuscular mycorrhizal fungi (AMF) was investigated in TM contaminated and long-term N fertilized soils. In addition, the role of different AMF species in TM uptake or sequestration was investigated. In the first experiment, AMF community structure was analyzed from non-contaminated and TM contaminated sites. We found the diversity of native AMF was lower in soils and plant roots harvested from TM polluted soils than from unpolluted soils. We also found that the community structure of AMF was modified by TM contamination. Some AMF ribotypes were more often associated with TM contaminated sites, other ribotypes with uncontaminated sites, while still other ribotypes were found both in polluted and unpolluted soils. In the second experiment, the effect of different organic and mineral N fertilization on AMF community structure was investigated. Variation in root-colonizing AMF community structure was observed in both organic and mineral fertilization. Some AMF ribotypes were more affiliated to organic fertilization and other to mineral fertilization. In addition, mineral fertilization reduced AMF ribotypes number while organic fertilization increased AMF ribotypes number. In this experiment, it was demonstrated that change in root-colonizing AMF community structure had a significant effect on plant productivity. In the third experiment, the role of different AMF species (G. irregulare and G. mosseae) in TM uptake by sunflower plants grown in soil amended with three different Cd levels was evaluated. It was demonstrated that AMF species differentially affected TM uptake or sequestration by sunflower plants. This experiment supported a different effect of AMF in TM uptake based on Cd concentration in soil and the AMF species involved. Our research demonstrated that TM and N fertilization affected and shifted AMF community structure within roots and soils. It was shown that change in root-colonizing AMF community structure significantly affected plant productivity. In this study, it was showed that the AMF species G. irregulare was recorded in all uncontaminated sites while G. mosseae was the most abundant AMF species in TM contaminated sites. Therefore, the role of G. irregulare and G. mosseae in Cd uptake by sunflower plants grown in soils amended with three different Cd levels was investigated. The results indicated that AMF species mediate different mechanisms to alleviate TM toxicity in host plants, depending on AMF species and soil Cd level involved. We hypothesize that G. irregulare is a potentially important species for Cd phytoextration processes, while G. mosseae might be a suitable candidate for Cd and Zn phytostabilization processes.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Pas de résumé en français / In plants, long distance transport of sugars from photosynthetic source leaves to sink organs comprises different crucial steps depending on the species and organ types. Sucrose, the main carbohydrate for long distance transport is synthesized in the mesophyll and then loaded into the phloem. After long distance transport through the phloem vessels, sucrose is finally unloaded towards sink organs. Alternatively, sugar can also be transferred to non‐plant sinks and plant colonization by heterotrophic organisms increases the sink strength and creates an additional sugar demand for the host plant. These sugar fluxes are coordinated by transport systems. Main sugar transporters in plants comprise sucrose (SUTs) and monosaccharide (MSTs) transporters which constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Although complete SUTs and MSTs gene families have been identified from the reference Dicot Arabidopsis thaliana and Monocot rice (Oriza sativa), sugar transporter families of the leguminous plant Medicago truncatula, which represents a widely used model for studying plant-fungal interactions in arbuscular mycorrhiza (AM), have not yet been investigated.With the recent completion of the M. truncatula genome sequencing as well as the release of transcriptomic databases, monosaccharide and sucrose transporter families of M. truncatula were identified and now comprise 62 MtMSTs and 6 MtSUTs. I focused on the study of the newly identified MtSUTs at a full family scale; phylogenetic analyses showed that the 6 members of the MtSUT family distributed in all three Dicotyledonous SUT clades; they were named upon phylogenetic grouping into particular clades: MtSUT1-1, MtSUT1-2, MtSUT1-3, MtSUT2, MtSUT4-1 and MtSUT4-2. Functional analyses by yeast complementation and expression profiles obtained by quantitative RT-PCR revealed that MtSUT1-1 and MtSUT4-1 are H+/sucrose symporters and represent key members of the MtSUT family. Conservation of transport capacity between orthologous leguminous proteins, expression profiles and subcellular localization compared to previously characterized plant SUTs indicate that MtSUT1-1 is the main protein involved in phloem loading in source leaves whilst MtSUT4-1 mediates vacuolar sucrose export for remobilization of intracellular reserve.The AM symbiosis between plants and fungi from the phylum Glomeromycota is characterized by trophic exchanges between the two partners. The fungus supplies the autotrophic host with nutrients and thereby promotes plant growth. In return, the host plant provides photosynthate (sugars) to the heterotrophic symbiont. Here, sugar fluxes from plant source leaves towards colonized sink roots in the association between the model leguminous plant M. truncatula and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices were investigated. Sugar transporter candidates from both the plant and fungal partners presenting differential expression profiles using available transcriptomic tools were pinpointed. Gene expression profiles of MtSUTs and sugar quantification analyses upon high and low phosphorus nutrient supply and inoculation by the AMF suggest a mycorrhiza-driven stronger sink in AM roots with a fine-tuning regulation of MtSUT gene expression. Conserved regulation patterns were observed for orthologous SUTs in response to colonization by glomeromycotan fungi.In parallel, a non-targeted strategy consisting in the development of a M. truncatula - G. intraradices expression library suitable for yeast functional complementation and screening of symbiotic marker genes, similar to the approach that led to the identification of the first glomeromycotan hexose transporter (GpMST1), has been developed in this study. [...]

La phytostabilisation est une méthode de gestion de sites pollués par des éléments traces métalliques (ETM) grâce à une approche verte. Les champignons mycorhiziens, associés aux racines, peuvent contribuer au potentiel de phytostabilisation des arbres en réduisant notamment le transfert des ETM vers les parties aériennes. Dans le cadre du projet BIOFILTREE, plusieurs approches sont mises en oeuvre afin de sélectionner des espèces fongiques performantes en phytostabilisation. Tout d'abord, afin de cibler des espèces d'intérêt, les communautés fongiques associées aux racines de divers clones de peupliers sur un site pollué sont analysées. Tous les peupliers sont colonisés par des champignons ecto-, endomycorhiziens et endophytes. Cependant, des spécificités d'hôtes sont observées parmi ces communautés, avec notamment un champignon majoritaire du genre Hebeloma qui ne s'associe pas avec tous les génotypes. Ensuite, plusieurs campagnes de prélèvements de champignons sur divers sites pollués ont permis d'isoler des souches particulièrement tolérantes aux ETM. De très fortes variations intra- et interspécifiques sont observées lors de ces tests. En parallèle, des approches ciblées de caractérisation fonctionnelle de gènes impliqués dans l'homéostasie au zinc sont mises en oeuvre chez Laccaria bicolor, un champignon ectomycorhizien modèle. L'homéostasie aux ETM est liée entre autres à l'activité des transporteurs des familles ZIP (Zrt-, Irt- related Protein) et CDF (Cation Diffusion Facilitator). L'étude des membres de ces deux familles a permis d'affiner la compréhension des mécanismes d'absorption et de séquestration des ETM dans les hyphes des champignons ectomycorhiziens / Phytostabilization is a gentle management option for sites polluted by trace elements (TE). Mycorrhizal fungi could assist plants in stabilizing pollutants by increasing the soil-prospected volume and by immobilizing MTE in their hyphae. Within the BIOFILTREE project, several approaches were used to select fungal strains that could be used for enhancing the phytostabilization process. Firstly, the mycorrhizal status of roots of three poplar clones from a TE-polluted site and the fungi associated with the roots were analyzed. The roots were colonized by endomycorrhizal, ectomycorrhizal, and endophytic fungi. Our data also revealed some specific trends, i.e. Hebeloma species was not associated with all poplar genotypes. Secondly, several fungal strains were isolated from polluted sites and their in vitro tolerance to TE was tested. There was a strong inter- and intra-specific variation in metal tolerance. In a greenhouse study, two poplar clones were inoculated with an endomycorrhizal inoculum and grown on a TE-polluted soil. A slight modification in TE accumulation in shoots was observed. In parallel, the role of ZIP (Zrt-Irt- like Proteins) and CDF (Cation Diffusion Facilitator) proteins in TE homeostasis/tolerance was also studied in the ectomycorrhizal model fungus Laccaria bicolor. The corresponding proteins were functionally characterized by the use of different approaches (yeast complementation, GFP-chimeric proteins, transcript analyses, cell metal content analyses). This study allowed us to better understand the mechanisms underlying zinc uptake and compartmentation in the hyphae of this fungus

Sustainable agriculture practices include a better exploitation of soil natural resources. Arbuscular mycorrhizal fungi (AMF) plays an important role in this context, with native communities in soils that establishes a mutualistic association with most plant species and maximizes their access to mineral nutrients, especially phosphorus. Seed treatment may consist in a good alternative to stimulate AMF root colonization, since early stages of crop growth, by using biostimulant formulations applied to seeds. This research had the objective to evaluate the early mycorrhization of soybean plants in response to seaweed extracts applied to seeds. The treatments consisted of a control (CTR, distilled water), two seaweed extracts, from Ascophyllum nodosum (ANE) and Ecklonia maxima (EME) species and a synthetic formulation containing formononetin (FOR), commercially used as mycorrhization stimulant. The first experiment was conducted in greenhouse, with the aim to evaluate different parameters of soybean early growth and development and AMF root colonization in response to the seed treatments, under different rates of phosphorus added to the soil (0, 50, 100 and 200 mg.dm-3). The second experiment was conducted in growth chamber, with the objective to evaluate qualitative and quantitative aspects of mycorrhization in response to the seed treatments with biostimulants, at early plant growth stages. In this experiment, the influence of treatments on the structure of AMF, bacterial and fungal communities in the soybean rhizosphere were analyzed, through PCR/T- RFLP fingerprinting. Phosphorus rates applied to soil significantly influenced (P<0.10) biometric and physiological parameters of soybean plants, while the seed treatments only influenced leaf chlorophyll content and nitrogen balance at a significant level, despite some trends to increase leaf area, shoot dry mass and nodulation. Formononetin applied to soybean seeds increases AMF root colonization compared to control, independently of P rate applied to soil, while ANE and EME presented intermediate values for this parameter, not differing from both control and formononetin. When considered some qualitative parameters of mycorrhiza establishment, such as frequency and intensity of mycorrhization, both synthetic and natural biostimulants presented significant improvements, with EME and FOR providing higher values in general. The phenological stage of soybean significantly influenced AMF, bacterial and fungal community structures in rizosphere, while the biostimulant seed treatments only significantly influenced the bacterial community. However, a relatively large variation in the microbial community structures could be attributed to the interaction between phenological stage of plants and the seed treatment with biostimulants. / Práticas agrícolas sustentáveis incluem uma melhor exploração dos recursos naturais do solo. Os fungos micorrízicos arbusculares (FMA) desempenham um papel importante neste contexto, com comunidades nativas nos solos que estabelecem uma associação mutualística com a maioria das espécies cultivadas e maximizam o acesso a nutrientes minerais, especialmente o fósforo. O tratamento de sementes pode consistir em uma alternativa interessante para estimular a colonização por FMA, desde estádios iniciais de crescimento da cultura, utilizando formulações de bioestimulantes aplicadas às sementes. Esta pesquisa teve como objetivo avaliar a micorrização precoce de plantas de soja em resposta a extratos de algas aplicados nas sementes. Os tratamentos consistiram de uma testemunha (CTR, água destilada), dois extratos de algas marinhas, das espécies Ascophyllum nodosum (ANE) e Ecklonia máxima (EME) e uma formulação sintética contendo formononetina (FOR), comercialmente utilizada como estimulante de micorrização. O primeiro experimento foi conduzido em casa de vegetação, com o objetivo de avaliar diferentes parâmetros de crescimento e desenvolvimento inicial das plantas de soja e a colonização de raízes por FMA, em resposta aos tratamentos de sementes; estas avaliações foram realizadas sob diferentes doses de fósforo adicionadas ao solo (0, 50, 100 e 200 mg.dm-3). O segundo experimento foi conduzido em câmara de crescimento, com o objetivo de avaliar aspectos qualitativos e quantitativos da micorrização em resposta ao tratamento de sementes com bioestimulantes, nas fases iniciais de crescimento das plantas. Neste experimento, analisou-se a influência dos tratamentos na estrutura de comunidades de FMA, bactérias e fungos na rizosfera das plantas de soja, através de PCR/T-RFLP. As doses de fósforo aplicadas no solo influenciaram significativamente (P<0,10) nos parâmetros biométricos e fisiológicos das plantas de soja, enquanto os tratamentos de sementes influenciaram significativamente apenas o teor de clorofila e o balanço de nitrogênio. A formononetina aplicada nas sementes de soja aumentou a colonização da raiz por FMA em relação ao controle, independentemente da dose de P aplicada ao solo, enquanto ANE e EME apresentaram valores intermediários para este parâmetro, não diferindo tanto da testemunha como da formononetina. Quando considerados alguns parâmetros qualitativos do estabelecimento de micorrizas, tanto os bioestimulantes sintéticos como os naturais apresentaram melhorias significativas. O estádio fenológico da soja influenciou significativamente as estruturas de comunidades de FMA, bactérias e fungos na rizosfera das plantas de soja, enquanto os tratamentos de sementes com bioestimulantes somente influenciaram significativamente a comunidade bacteriana. No entanto, uma variação relativamente grande nas estruturas da comunidade microbiana pôde ser atribuída à interação entre estádio fenológico das plantas e o tratamento de sementes com bioestimulantes.

A conservação da biodiversidade edáfica na Floresta Ombrófila Mista favorece os processos de decomposição e ciclagem de nutrientes, melhorando as propriedades físicoquímicas e biológicas do solo. O objetivo deste trabalho foi verificar o potencial dos atributos da macrofauna edáfica, Fungos Micorrízicos Arbusculares (FMA) e de variáveis ambientais explicativas (físico-químicas e microbiológicas) como indicadores de qualidade do solo em duas épocas contrastantes (inverno e verão) em Floresta de Araucária do Estado de São Paulo. Foram avaliadas florestas com Araucaria angustifolia nativa (NF) e reflorestada (RF) em três regiões distintas do Estado de São Paulo. Cada floresta teve três repetições verdadeiras, nas quais, foram coletadas, ao acaso, quinze amostras de solo para avaliação dos atributos físicos (densidade, macroporosidade, microporosidade, porosidade total e umidade), químicos [pH, teores de carbono orgânico (C-org), P, Ca, Mg, K, Al e H+Al] e biológicos [macrofauna, FMA, carbono da biomassa microbiana (CBM), respiração basal, quociente metabólico (qCO2) e atividade da desidrogenase (Desi)]. Nos mesmos pontos de amostragem, foram avaliadas a macrofauna edáfica, a densidade de FMA e a colonização micorrízica em raízes de araucária. Na serapilheira foi avaliada a massa seca (MSS) e foram determinados os teores de C, N e S. Os atributos físico-químicos e microbiológicos do solo e químicos da serapilheira foram submetidos à análise de variância (Two-way ANOVA). A abundância da macrofauna e de espécies de minhocas foram submetidas à Análise de Componentes Principais (ACP) e as variáveis microbiológicas, MSS, físico-químicas do solo e da serapilheira foram usadas como variáveis explicativas (VE). A abundância de espécies de FMA foi submetida à ACP e as variáveis físico-químicas do solo e da serapilheira foram usadas como VE. Adicionalmente, a abundância da macrofauna e as VE foram submetidas à Análise Canônica Discriminante (ACD) e a uma Análise de Correlação Canônica (ACC). A macrofauna apresentou maior riqueza de grupos no verão, sendo que a NF proporcionou a maior diversidade de grupos. A umidade, porosidade total, teor de S na serapilheira, pH, teor de K, CBM e respiração basal foram os atributos edáficos mais importantes para separar as áreas. O grupo Oligochaeta, principamente as espécies Ponthoscolex corethrurus e Amynthas spp., foram boas indicadoras de qualidade do solo e/ou de distúrbio na Floresta de Araucária. Encontraram-se 36 táxons de FMA, sendo os gêneros Glomus, Acaulospora e Scutellospora os mais abundantes. A espécie Ambispora appendicula apresentou a maior abundância relativa. No inverno, observou-se alta correlação da Desi e C-CO2 com pH e H+Al, enquanto que no verão, o C-org, umidade, Mg, pH e C da serapilheira se correlacionaram com a atividade da Desi e C-CO2. Os atributos H+Al, P, porosidade total, S da serapilheira e umidade do solo foram os mais importantes na separação das áreas, independente de épocas de coleta. / The conservation of edaphic biodiversity in Araucaria Forests favors the processes of decomposition and nutrient cycling, improving the physico-chemical and biological soil properties. The objective of this study was to investigate the potential of the macrofauna, mycorrhizal fungi (AMF) and environmental explanatory variables (physico, chemical and microbiological) as indicators of soil quality in two different contrasting seasons (winter and summer) in Araucaria Forests. We evaluated forests with native Araucaria angustifolia (NF) and reforested areas (RF) in three different regions in the State of São Paulo. Each forest had three true replicates, in which we collected at random, fifteen soil samples for evaluation of physical attributes (density, macroporosity, microporosity, total porosity and moisture), chemical attributes [pH, organic carbon (org-C), P, Ca, Mg, K, Al and H+Al] and biological attributes [macrofauna, AMF, microbial biomass carbon (MBC), basal respiration (CO2-C), metabolic quotient (qCO2) and dehydrogenase activity (Desi)]. At the same sampling points we evaluated the edaphic macrofauna, the density of AMF and root colonization in Araucaria. We evaluated the litter dry matter (MSS) and analyzed the contents of C, N and S. The physico-chemical and microbiological soil attributes and the chemical litter attributes were submitted to analysis of variance (Two-way ANOVA). The abundance of macrofauna and of earthworm species were submitted to a Principal Components Analysis (PCA) and the microbiological variables, litter dry weight, physico-chemical attributes of soil and litter were used as explanatory variables (EV). The abundance of AMF species was submitted to the PCA and the physico-chemical variables of soil and litter were used as EV. Additionally, the abundance of macrofauna and EV were submitted to a Canonical Discriminant Analysis (CDA) and to one Canonical Correlation Analysis (CCA). The macrofauna showed greater richness of groups in the summer, and the NF provided the highest diversity. Moisture, total porosity, S litter content, pH, K, MBC and basal respiration were the edaphic attributes most important to separate the areas. The group Oligochaeta, especially the species Ponthoscolex corethrurus and Amynthas spp., were good indicators of soil quality and/or disturbance in these Araucaria forests. We found 35 AMF taxons, and the genera Glomus, Acaulospora and Scutellospora were the most abundant. The species Ambispora appendicula showed the highest relative abundance. In winter, a high correlation of the microbiological attributes Desi and CO2-C with pH and H+Al was found, while in the summer, the org-C, moisture, Mg, pH and Carbon of the litter were more correlated with Desi activity and C-CO2. The attributes H+Al, P, total porosity, S of the litter and soil moisture were the most important attributes to discriminate the areas, regardless of the season.

A degradação de extensas áreas devido às atividades mineradoras a céu aberto tem trazido sérios prejuízos econômicos e, principalmente, graves danos ao ambiente. Esse tipo de atividade, além de alterar as características originais dos solos, levam a perda de grande parte de seus conteúdos de matéria orgânica e da biodiversidade natural, condições básicas para uma atividade biológica sustentável. A revegetação dos solos dessas áreas, com o objetivo de restabelecer suas características químicas, físicas e biológicas a um nível mínimo que permita o desenvolvimento de espécies vegetais e da atividade microbiana, tão importante no estabelecimento e sucessão da macrobiota, constitui-se numa prática muito recomendada. O objetivo dessa tese foi avaliar o potencial de reabilitação do solo degradado de uma área de mineração de cassiterita na região da Floresta Nacional do Jamari-RO, através de ensaios experimentais em casa-devegetação, utilizando-se a revegetação do rejeito com seis espécies de crescimento rápido e o manejo de microrganismos simbiontes fixadores do N2 e fungos micorrízicos arbusculares, associados ou não à adubação com composto orgânico e termofosfato. As espécies avaliadas foram Canavalia ensiformis, Cajanus cajan, Acacia mangium, Parkia multijuga, Schinus terebinthifolia e Inga edulis. Em casa-de-vegetação, os ensaios mostraram efeito significativo da aplicação do composto orgânico para todas as espécies cultivadas. A associação da adubação orgânica com a inoculação micorrízica produziu maiores incrementos no peso da matéria seca da parte aérea de C. cajan, A. mangium e S. terebinthifolia do que a adubação com composto orgânico sem inoculação micorrízica. As leguminosas nodulíferas C. cajan e A. mangium também produziram mais matéria seca de nódulos quando associadas à adubação orgânica e fungos micorrízicos. À exceção de P. multijuga, a acumulação de N e P na parte aérea foi aumentada na presença do composto orgânico em todas as espécies porém, em C. cajan, A. mangium e S. terebinthifolia, esse acúmulo foi maior nas plantas micorrizadas. Na ausência de adubação com composto orgânico algumas espécies micorrizadas mostraram maior eficiência na utilização do termofosfato. O pH do rejeito após cultivo foi sempre maior do que aquele do rejeito original. Entretanto, nos vasos que continham plantas inoculadas com FMA e adubadas com composto orgânico, este incremento foi menor. A inoculação com FMA contribuiu para o aumento da quantidade de micélio externo total para a maioria das espécies cultivadas no rejeito. Pelos resultados obtidos, podemos concluir que, para o sucesso da revegetação do rejeito de mineração de cassiterita, a adição de composto orgânico é essencial para o estabelecimento das plantas e da microbiota do solo. As plantas inoculadas com fungos micorrízicos e adubadas com composto orgânico se desenvolvem melhor no rejeito do que as plantas não micorrizadas. As espécies vegetais devem ser selecionadas preferencialmente entre as micotróficas e as nodulíferas, uma vez que podem se estabelecer no ambiente com um menor aporte de insumos e com práticas de manejo menos onerosas. / The increasing degraded soil areas caused by open cast mining activities has brought critical damages to the environment. The mine spoil must be ameliorated with anthropogenic interferences, consisting of revegetating soils, introducing organic matter and microbial diversity, basic conditions for a sustainable soil biological activity. The objective of this thesis was to evaluate the rehabilitation potential of a mine spoil area by cultivating fast growing plant species and inoculating rhizobia and arbuscular mycorrhizal fungi, in the presense or absence of organic compost or thermophosphate. Thus, six plant species were cultivated under green-house conditions, to evaluate the potential of plant establishment on a cassiterite mining waste. Shoot height and dry weight, substrate pH, N and P shoot content, external mycelium and root nodule numbers and dry weight were determined. Fertilization with organic compost and mycorrhizal inoculation improved shoot dry weight of C. cajan, A. mangium and S. terebinthifolia and root nodule dry weight of the nodulating legumes. N and P shoot contents were higher in plants that were inoculated with arbuscular mycorrhizal fungi and fertilized with organic compost. The soil pH after cultivation was always much higher than the initial pH, somewhat in the presense of organic compost and mycorrhizal inoculation of plants. External mycelium in the mining waste increased with mycorrhizal inoculation. We can conclude that for a successful revegetation of cassiterite mining spoil the addition of organic matter is essencial for plant and microrganism establishment. It provides a better nutrient cycling and improves chemical and physical substrate characteristics. Plants inoculated with mycorrhizal fungi and growing in the presense of compost developded much better than non-mycorrhizal plants. Species selected for revegetation should be preferencially mycotrohphic and nodulating legumes as they will grow well even without the use of expensive fertilizers and management practices.

O objetivo deste trabalho foi testar um manejo Alternativo em um pomar convencional de Tangerina Murcote (Citrus reticulata, Blanco X Citrus sinensis, Osbeck) enxertadas sobre limão cravo (Citrus limonia, Osbeck), com utilização de composto fermentado Bokashi, avaliando seu efeito sobre a compactação do solo, colonização radicular de fungos micorrízico arbusculares (FMA) nativos, desempenho nutricional, produção e as características tecnológicas dos frutos. O experimento foi delineado separando-se um hectare dentro de um talhão em produção, com 10 anos de idade, onde foram suspensas as aplicações de fertilizantes e defensivos químicos, inclusive os herbicidas, substituindo-os por fontes de nutrientes de baixa solubilidade, de forma que viessem incentivar as interações biológicas, em especial as associações micorrízicas. Para estimular a cobertura vegetal, foi aplicado o composto fermentado Bokashi sobre a vegetação espontânea da linha e entrelinha, procedendo-se o roço mecânico, cuja área foi denominada ‘tratamento Alternativo’. Uma área equivalente e contígua, onde os tratos culturais e fitossanitários convencionais foram mantidos, foi utilizada para fins de comparação, sendo esta denominada ‘tratamento Convencional’. Os parâmetros eleitos para comparação foram: resistência do solo à penetração, densidade radicular superficial, colonização de fungos micorrízicos arbusculares nativos, assim como a quantidade de esporos viáveis no solo, análise de conteúdo de nutriente foliar e produtividade e qualidade de fruto, sendo avaliadas 10 parcelas de 3 plantas para cada tratamento. O manejo Alternativo, com aplicação de composto fermentado Bokashi sobre a vegetação espontânea na entrelinha, propiciou maior cobertura vegetal, 107,5% a mais, avaliado em janeiro de 2005, principalmente de gramíneas, a qual reduziu a resistência do solo à penetração ao longo do experimento, denotando melhora no estado de compactação do solo em relação ao tratamento Convencional. Também, houve melhor desenvolvimento do sistema radicular superficial, maior índice de colonização radicular de fungos micorrízicos arbusculares (FMA) nativos nas plantas do tangor ‘Murcott’, em média 10% superior, e em conseqüência, maior quantidade de esporos viáveis no solo, sendo em média 23% superior ao tratamento Convencional. Em termos nutricionais, a quantidade aportada de K no tratamento Alternativo não foi suficiente para suprir adequadamente as necessidades das plantas, demonstrando que é preciso dar maior atenção a esse nutriente nas propostas alternativas de manejo para citros. O teor de B foliar se apresentou significativamente superior e em níveis crescentes ao longo do experimento, atingindo patamares 280% maiores do que o tratamento Convencional, com teores de até 161,35 mg.kg-1, resultado altamente desejável para este nutriente em citros. Apesar da redução na produtividade inicialmente, o tratamento Alternativo foi superior em massa de frutos por planta na última avaliação, em agosto de 2004, sendo 58,2 kg. planta-1 contra 42,9 kg.planta-1 do Convencional, embora o tamanho dos frutos e a acidez total tenham sido prejudicados pela deficiência do K. Isso demonstra que em pomar de idade avançada o manejo alternativo deve ser adotado de forma gradual. Em avaliações complementares foi observado que o manejo Alternativo proporcionou melhor equilíbrio na população de Orthezia praelonga, tendo 0,54% das plantas atacadas, contra 14,06% no manejo Convencional. / The objective of this study was to test an alternative management in a conventional orchard of ‘Murcott’ tangor (Citrus reticulata, Blanco X Citrus sinensis, Osbeck) grafted on rangpur lime (Citrus limonia, Osbeck) using fermented compost Bokashi, in order to evaluate its effect on soil decompaction, root colonization of indigenous arbuscular mycorrhizal fungi (AMF), nutritional performance, yield and technological characteristics of fruits. The experiment was set by separating one hectare plot in a ten year old yielding orchard, where chemicals fertilizers and pesticides, including herbicides, were discontinued and replacing them by sources of low solubility nutrients and alternative pest control, with the purpose to encouraging the biological interactions, mainly mycorrhizal associations. To stimulate vegetal covering, a fermented compost Bokashi was applied on the spontaneous vegetation of the row e interrow, mechanical clearing was used, and the area was designated ‘alternative treatment’. An equivalent and adjoining area, where conventional cultural and phythosanitary treatments were maintained was used for comparative purposes, and was designated ‘conventional treatment’. The parameters chosen for comparison were: soil resistance to penetration, surface root density, colonization of indigenous arbuscular mycorrhizal fungi, as well as amount of viable spores in the soil, analysis of foliar nutrient content, and fruit yield and quality. Ten plots of 3 plants per each treatment were evaluated. The alternative management with application of fermented compost Bokashi on the spontaneous vegetation on the interrow provided a 107.5% increase in vegetal covering, mainly grasses, measured in January 2005, which decreased soil resistance to penetration and resulted in an improvement in soil compaction state as compared with the conventional treatment. Also, there were an improved development of surface root systems, a higher rate of root colonization of indigenous arbuscular mycorrhizal fungi (AMF) in plants of ‘Murcott’ tangor, averaging 10% higher and, consequently, a higher amount of viable spores in the soil, an average of 23% higher than of the conventional treatment. In nutritional terms, the input of K in the alternative treatment was not enough to supply adequately the needs of the plants, indicating that more attention is required in relation to this nutrient in alternative proposals of citrus management. The foliar content of B was shown to be significantly higher and at increasing rates along the experiment, reaching levels 280% higher than those of the conventional treatment, having reached contents of up to 161.35 mg.kg-1, a highly desirable result for citrus nutrition. Despite the initial yield decrease, the alternative treatment showed a higher fruit mass per plant in the last evaluation in August 2004, i.e. 58.2 kg.plant-1 against 42.9 kg.plant-1 in the conventional treatment, even though fruit size and total acidity were impaired by K deficiency. This shows that, in older aged orchard, an alternative management should be introduced gradually. Subsequent appraisals showed that the alternative treatment provide a better equilibrium in the population of Orthezia praelonga, i.e. 0.54% of attacked plants against 14.06% in the conventional treatment.

Os hormônios vegetais etileno e ABA são possíveis reguladores chave do desenvolvimento das micorrizas arbusculares (MAs). O primeiro objetivo deste trabalho foi avaliar o desenvolvimento da MA em mutantes superprodutores e insensíveis ao etileno (epinastic e Never ripe, respectivamente) e verificar se enzimas relacionadas ao sistema de defesa vegetal são reguladas diferencialmente pelo etileno durante o desenvolvimento da simbiose A colonização de raízes de epinastic (epi) por G. clarum foi significativamente reduzida quando comparada com Micro-Tom (MT), além disso, a funcionalidade da simbiose, demonstrada pelo acúmulo de transcritos de LePT4, foi fortemente reduzida quando comparada com MT. Por outro lado, o padrão de colonização e funcionalidade da simbiose no mutante Nr não diferiu de MT. Indução no acúmulo de transcritos de genes que codificam quitinases e enzimas relacionadas ao estresse oxidativo foi observada no mutante epi, enquanto o acúmulo de transcritos de uma -1,3-glucanase foi fortemente induzido no mutante Nr. O segundo objetivo foi verificar se a colonização reduzida observada em mutantes deficientes em ABA é dependente da produção do etileno. Para tanto, mutantes que diferem na deficiência de ABA e produzem diferentes quantidades de etileno (notabilis e sitiens) e o mutante epi foram inoculados com G. clarum e tratados com aminoethoxivinilglicina (AVG), um inibidor de etileno. A colonização intrarradicular de G. clarum nos mutantes epi e notabilis (not) foi significativamente reduzida quando comparada com MT, sendo que o mutante sitiens (sit) foi o menos susceptível à colonização em relação aos demais genótipos. A aplicação de AVG a 10 µM restaurou completamente a colonização em epi, mas não nos mutantes deficientes em ABA. O acúmulo de transcritos de LePT4 confirmou os resultados de colonização. Os genes que codificam enzimas chave na biossíntese do etileno, LeACS2 e LeACO4, foram regulados positivamente pela inoculação com G. clarum, e a restauração do desenvolvimento da MA observada em epi que recebeu AVG, provavelmente envolveu ACC oxidase (LeACO4). O acúmulo de transcritos dos genes que codificam ACC oxidase (LeACO1 e LeACO4) foi induzido significativamente em sit, mostrando que a superprodução de etileno observada neste mutante pode envolver a regulação da ACC oxidase. Por fim, indução no acúmulo de transcritos do gene LeNCED foi observada em todos os genótipos micorrizados e em raízes de epi que receberam AVG, demonstrando que LeNCED é regulado positivamente pela inoculação com G. clarum e que o etileno regula a expressão deste gene. Os resultados demonstram que etileno e ABA estão envolvidos na regulação do desenvolvimento de MA e que etileno modula a expressão de genes relacionados ao sistema de defesa vegetal permitindo o estabelecimento de uma simbiose funcional. / The plant hormones ethylene and ABA are probably key developmental regulators of arbuscular mycorrhizae (AM). The first objective of this work was to evaluate the development of MA in overproducing and insensitive mutants to ethylene (epinastic and Never ripe, respectively) and verify if enzymes related to plant defense system are differentially regulated by ethylene during the symbiosis development. The colonization of epinastic (epi) roots by G. clarum was significantly reduced when compared with Micro-Tom (MT), moreover, the functionality of the symbiosis as demonstrated by transcripts accumulation of LePT4, was strongly reduced when compared with MT. Still, the pattern of colonization and functionality of the symbiosis in the Nr mutant did not differ from MT. Induction in the transcripts accumulation of genes encoding chitinase and enzymes related to oxidative stress was observed in epi mutant, whereas transcripts accumulation of -1, 3-glucanase was strongly induced in the Nr mutant. The second objective was to determine whether the reduced colonization observed in mutants deficient in ABA is dependent on ethylene production. For this purpose, mutants that differ in ABA deficiency and produce different amounts of ethylene (notabilis and sitiens) and the epi mutant were inoculated with G. clarum and treated with an ethylene inhibitor called aminoethoxivinilglicin (AVG). The intraradical colonization of G. clarum in epi and notabilis (not) mutants was significantly reduced when compared with MT and sitiens mutant was less susceptible to colonization in comparison with other genotypes. The application of AVG to 10 µM completely restored the colonization in the epi but not in mutants deficient in ABA. The transcripts accumulation of LePT4 confirmed the results of colonization. The genes that encode key enzymes in the biosynthesis of ethylene, LeACS2 and LeACO4, were positively regulated by inoculation with G. clarum and the restoration in the development of MA observed in epi which received AVG probably involved ACC oxidase (LeACO4). Transcripts accumulation of genes that encode ACC oxidase (LeACO1 and LeACO4) were induced significantly in sit showing that overproduction of ethylene in this mutant can involve ACC oxidase. Finally, the induction in the accumulation of gene transcripts LeNCED was observed in all genotypes and mycorrhizal roots of epi which received AVG demonstrating that LeNCED is regulated positively by the inoculation with G. clarum and that ethylene regulates the expression of this gene. Results show that ethylene and ABA are involved in regulating of MA development and ethylene modulates the expression of genes related to the plant defense system allowing the establishment and functionality of the symbiosis.