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

The thesis deals with the effect of arbuscular mycorrhiza (AM) on the growth of selected plant species at a locality in České středohoří. This locality is characterized by close neighborhood of a semi-natural dry grassland and a former field abandonned in the 1990s, typical for the mosaic of biotopes in the region. The study is based on previous findings that some plant species, which are common at the semi-natural dry grasslands, do not colonize the abandoned fields. As AM is an important factor affecting diversity and productivity of plant communities we hypothesized that this phenomenon could be related to changes in AM fungal communities at the abandoned field. The hyphothesis was tested in a greenhouse pot experiment with three taxonomically related pairs of plant species, always one species growing at the abandoned field and the second one not. Growth and phosphorus uptake of the plants was followed in soils of both biotopes after factorial inoculation with AM fungal communities from both biotopes. The experiment was complemented by description of AM fungal communities in the roots of six plant species pairs from the locality using terminal restriction fragment length polymorphism (T-RFLP). The greenhouse experiment revealed positive mycorrhizal response in all plant species, but the origin...

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016. / South Africa, as one of the leaders in mining industry, due to the variety and quantity of minerals produced, has been and is still producing a number of mine tailings which are contaminated by heavy metals. Heavy metals are very harmful to plants and especially to human beings and animals due to their non-biodegradable nature. The problem of environmental metal pollution could be combated by the establishment of Arbuscular Mycorrhiza (AM) vegetation on the surface of mine tailings. Besides the toxicity of the substrate, such areas usually lack essential nutrients (mainly N, P, and K) and organic matter. AM fungi contribute to soil structure by forming micro- and macro- soil aggregates within the net of external hyphae. Their presence may reduce stress caused by lack of nutrients or organic matter and increase plant resistance to pathogens, drought and heavy metals. Therefore, mycorrhizal fungi may become the key factor in successful plant revegetation of heavy-metal-polluted areas by promoting the success of plant establishment and increasing soil fertility and quality. The aim of this project was to identify AM fungi from a number of heavy metal sites in South Africa using both morphological and molecular techniques, followed by the evaluation of heavy metal distribution and localisation in mycorrhizal roots. Soil samples were collected from three different provinces, namely: Gauteng, Mpumalanga and North West provinces. The sites were selected based on their historical and current heavy metal contamination. Indigenous AM fungal isolates (which are adapted to local soil conditions) can stimulate plant growth better than non-indigenous isolates. AM fungal spores were isolated from 100g of representative soil sample by the wet sieving and decanting method, followed by assessment of spore numbers and infective propagules. The spores of a subset of the pot samples were mounted on microscope slides in polyvinyl lactic acid glycerol and identified by morphological characteristics to the level of genus or species. Most of the spores counted were observed in a 45 μm sieve. These spores were tiny and had different sizes, colours and shapes. The majority of the observed spores were small, brown and oval in shape. For morphological identification, plant roots were stained and hyphae were found to be the most abundant in roots. For molecular identification, two sets of nested PCR primers, namely NS1 & NS4 coupled with AML1 & AML2, were employed in this study due to their ability to amplify all subgroups of arbuscular mycorrhizal fungi (AM fungal, Glomeromycota), while excluding sequences from other organisms. Through both morphological characteristics and molecular identification, the following fungal genera were identified for the first time in the studied sites in South Africa. The study identified a total of 14 AM fungal genera and 55 AM fungal species, which are: Glomus (15), Acaulospora (11), Scutellospora (6), Gigaspora (6), Rhizophagus (3), Funneliformis (3), Archaeospora (2), Claroideoglomus (2), Ambispora (2), Sclerocystis (1), Fuscutata (1), Entrophospora (1), Diversispora (1), Paraglomus (1). Both Glomus and Acualospora have been observed to be the highest occurring genera in the analysed soil samples, followed by Scutellospora and Gigaspora and others mentioned. PIXE technique was successful in localising elemental concentration in both plant roots and AM fungal structures, as well as in indicating the large vesicles in root tissue. AM fungal structures in the outer cortex or outer epidermal layer of the root cross-sections were observable, as shown by the more significantly enriched Si in the vesicles and arbuscules. Distinctive elemental maps can be used to localise sites of colonisation and verification of the symbiotic nature of the tissue. This indicates that a range of metals can be sequestered in AM fungal structures above levels in surrounding host root tissue, and demonstrates the potential of Micro-PIXE to determine metal accumulation and elemental distribution in mycorrhizal plant roots and inter-and intracellular AM fungal structures. This research highlights the potential of AM fungi for inoculation of plants as a prerequisite for successful restoration of heavy metal contaminated soils. It also illustrates the importance of AM fungal diversity in selected high heavy metal (HM) sites in RSA, particularly in the North West and the Gauteng gold mining slime dams. Therefore, phytoremediation of mine tailings by mycorrhizal plants seems to be one of the most promising lines of research on mine tailings contamination by heavy metals. The strategies which evolved during this project have great potential for phytoremediation of toxic mining sites, and thus can help mitigate the environmental problems, especially in the mining waste sites. / LG2017

Micorrizas arbusculares (MAs) são associações simbióticas entre os fungos do filo Glomeromycota e a maioria das plantas. Os mecanismos moleculares que controlam o processo de colonização e desenvolvimento das MAs são ainda pouco conhecidos, mas proteínas com acúmulo diferencial em MAs podem ter papel regulatório importante. O presente trabalho teve como objetivo detectar, por meio de eletroforese bi-dimensional (2D-PAGE) e espectrometria de massas, proteínas com acúmulo diferencial no fluido intercelular (FI), membrana plasmática ou tecido radicular de cana-de-açúcar colonizada por Glomus clarum. Plântulas de cana-de-açúcar micropropagadas foram inoculadas com G. clarum e cultivadas com 20 ou 200 mg de P kg-1 de substrato. Raízes micorrizadas e não-micorrizadas, 8 semanas após a inoculação, foram utilizadas para a extração de proteínas do FI, membrana plasmática e tecido radicular (solúveis totais). As proteínas foram separadas por 2D-PAGE e analisadas por espectrometria de massas. Os perfis de proteínas solúveis totais e de membrana plasmática não revelaram proteínas relacionadas à simbiose. No entanto, três proteínas do FI, uma hidrolase aspártica putativa, uma histidina quinase putativa e uma peroxidase putativa apresentaram acúmulo induzido em raízes micorrizadas. As atividades de peroxidases nas raízes e apoplasto das raízes foram determinadas. A atividade de peroxidase apoplástica foi maior em raízes colonizadas e cultivadas em baixo teor de P, quando comparado com controles não-inoculados. Com base na seqüência parcial de aminoácidos dessa peroxidase, um fragmento de seu gene (POX1) foi amplificado e clonado a partir de cDNA de raízes de cana-de-açúcar. A sequência obtida mostrou 90% e 91% de identidade com peroxidase de milho (NCBI) e cana-de-açúcar (TIGR), respectivamente. A análise de expressão de POX1 foi feita por PCR quantitativo a partir de transcritos extraídos de raízes micorrizadas e não-micorrizadas, em condições de baixo e alto P. O acúmulo de transcritos de POX1 em raízes micorrizadas em condições de baixo P foi 6,8 vezes maior do que em raízes micorrizadas cultivadas em condições de alto P. Raízes micorrizadas e cultivadas em condições de alto P apresentaram acúmulo de transcritos 3,9 vezes menor do que em raízes nãomicorrizadas cultivadas nas mesmas condições de P. Os dados obtidos sugerem que o controle do metabolismo de espécies ativas de oxigênio é um dos fatores que contribuem para a regulação do desenvolvimento de MAs. Estudos com plantas alteradas para a expressão de POX1 são, no entanto, necessários para elucidar a essencialidade dessa peroxidase nas MAs. / Arbuscular mycorrhizae (AM) are symbiotic associations between fungi of the phylum Glomeromycota and most of the plant species. Even though the molecular mechanisms controlling the colonization process and AM development are largely unknown, proteins with differential accumulation in AM may have important regulatory roles. The aim of this work was to detect, by bi-dimensional electrophoresis (2D-PAGE) and mass spectrometry, proteins with differential accumulation in the intercellular fluid (IF), plasma membrane or radicular tissue of sugarcane colonized by Glomus clarum Micropropagated sugarcane plantlets were inoculated with G. clarum and growth under low or high P conditions, 20 or 200 mg P kg-1 substrate, respectively. Mycorrhizal and non-mycorrhizal roots, eight weeks after inoculation, were used to extract proteins from the IF, plasma membrane and root tissue (total soluble proteins). Protein separation and analyses were performed using 2D-PAGE and mass spectrometry. The total soluble and plasma membrane protein profiles did not reveled symbiosis-related proteins. However, three proteins from the IF, a putative aspartic hydrolase, a putative histidine kinase and a putative peroxidase showed induced accumulation in mycorrhizal roots. Peroxidase activities in roots and apoplastic fluid were determined, and shown to be higher in mycorrhizal roots at low P than in non-mycorrhizal control roots. Based on the partial amino acid sequence of this peroxidase, a partial cDNA sequence of its gene (POX1) was cloned from PCR-amplified cDNA from sugarcane roots. The POX1 sequence showed 90% and 91% identity to maize (NCBI) and sugarcane (TIGR) peroxidase, respectively. Expression analyses of POX1 were perfomed using quantitative PCR of reverse transcripts from mycorrhizal and non-mycorrhizal roots at low and high P conditions. The steady state level of POX1 transcripts in mycorrhizal roots at low P condition was 6.8-fold higher than in mycorrhizal roots at high P conditions. In mycorrhizal roots at high P conditions the steady state level of POX1 transcripts was 3.9-fold lower than in nonmycorrhizal control roots. These data suggest that the metabolism of reactive oxygen species may be an important factor controlling the development of AM. Studies with plants altered in POX1 expression are, however, required to elucidate the essentiality of this peroxidase in AM.

Micorrizas arbusculares (MAs) são associações simbióticas entre os fungos do filo Glomeromycota e a maioria das plantas. Os mecanismos moleculares que controlam o processo de colonização e desenvolvimento das MAs são ainda pouco conhecidos, mas proteínas com acúmulo diferencial em MAs podem ter papel regulatório importante. O presente trabalho teve como objetivo detectar, por meio de eletroforese bi-dimensional (2D-PAGE) e espectrometria de massas, proteínas com acúmulo diferencial no fluido intercelular (FI), membrana plasmática ou tecido radicular de cana-de-açúcar colonizada por Glomus clarum. Plântulas de cana-de-açúcar micropropagadas foram inoculadas com G. clarum e cultivadas com 20 ou 200 mg de P kg-1 de substrato. Raízes micorrizadas e não-micorrizadas, 8 semanas após a inoculação, foram utilizadas para a extração de proteínas do FI, membrana plasmática e tecido radicular (solúveis totais). As proteínas foram separadas por 2D-PAGE e analisadas por espectrometria de massas. Os perfis de proteínas solúveis totais e de membrana plasmática não revelaram proteínas relacionadas à simbiose. No entanto, três proteínas do FI, uma hidrolase aspártica putativa, uma histidina quinase putativa e uma peroxidase putativa apresentaram acúmulo induzido em raízes micorrizadas. As atividades de peroxidases nas raízes e apoplasto das raízes foram determinadas. A atividade de peroxidase apoplástica foi maior em raízes colonizadas e cultivadas em baixo teor de P, quando comparado com controles não-inoculados. Com base na seqüência parcial de aminoácidos dessa peroxidase, um fragmento de seu gene (POX1) foi amplificado e clonado a partir de cDNA de raízes de cana-de-açúcar. A sequência obtida mostrou 90% e 91% de identidade com peroxidase de milho (NCBI) e cana-de-açúcar (TIGR), respectivamente. A análise de expressão de POX1 foi feita por PCR quantitativo a partir de transcritos extraídos de raízes micorrizadas e não-micorrizadas, em condições de baixo e alto P. O acúmulo de transcritos de POX1 em raízes micorrizadas em condições de baixo P foi 6,8 vezes maior do que em raízes micorrizadas cultivadas em condições de alto P. Raízes micorrizadas e cultivadas em condições de alto P apresentaram acúmulo de transcritos 3,9 vezes menor do que em raízes nãomicorrizadas cultivadas nas mesmas condições de P. Os dados obtidos sugerem que o controle do metabolismo de espécies ativas de oxigênio é um dos fatores que contribuem para a regulação do desenvolvimento de MAs. Estudos com plantas alteradas para a expressão de POX1 são, no entanto, necessários para elucidar a essencialidade dessa peroxidase nas MAs. / Arbuscular mycorrhizae (AM) are symbiotic associations between fungi of the phylum Glomeromycota and most of the plant species. Even though the molecular mechanisms controlling the colonization process and AM development are largely unknown, proteins with differential accumulation in AM may have important regulatory roles. The aim of this work was to detect, by bi-dimensional electrophoresis (2D-PAGE) and mass spectrometry, proteins with differential accumulation in the intercellular fluid (IF), plasma membrane or radicular tissue of sugarcane colonized by Glomus clarum Micropropagated sugarcane plantlets were inoculated with G. clarum and growth under low or high P conditions, 20 or 200 mg P kg-1 substrate, respectively. Mycorrhizal and non-mycorrhizal roots, eight weeks after inoculation, were used to extract proteins from the IF, plasma membrane and root tissue (total soluble proteins). Protein separation and analyses were performed using 2D-PAGE and mass spectrometry. The total soluble and plasma membrane protein profiles did not reveled symbiosis-related proteins. However, three proteins from the IF, a putative aspartic hydrolase, a putative histidine kinase and a putative peroxidase showed induced accumulation in mycorrhizal roots. Peroxidase activities in roots and apoplastic fluid were determined, and shown to be higher in mycorrhizal roots at low P than in non-mycorrhizal control roots. Based on the partial amino acid sequence of this peroxidase, a partial cDNA sequence of its gene (POX1) was cloned from PCR-amplified cDNA from sugarcane roots. The POX1 sequence showed 90% and 91% identity to maize (NCBI) and sugarcane (TIGR) peroxidase, respectively. Expression analyses of POX1 were perfomed using quantitative PCR of reverse transcripts from mycorrhizal and non-mycorrhizal roots at low and high P conditions. The steady state level of POX1 transcripts in mycorrhizal roots at low P condition was 6.8-fold higher than in mycorrhizal roots at high P conditions. In mycorrhizal roots at high P conditions the steady state level of POX1 transcripts was 3.9-fold lower than in nonmycorrhizal control roots. These data suggest that the metabolism of reactive oxygen species may be an important factor controlling the development of AM. Studies with plants altered in POX1 expression are, however, required to elucidate the essentiality of this peroxidase in AM.

Orientador: Sidney Luiz Stürmer. / Co-orientador: Luis Hamilton Pospissil Garbossa. / Dissertação (Mestrado em Engenharia Ambiental) - Programa de Pós-Graduação em Engenharia Ambiental, Centro de Ciências Tecnológicas, Universidade Regional de Blumenau, Blumenau.

Orientador: Sidney Luiz Stürmer. / Dissertação (Mestrado em Engenharia Ambiental) - Programa de Pós-Graduação em Engenharia Ambiental, Centro de Ciências Tecnológicas, Universidade Regional de Blumenau, Blumenau.

As plantas apresentam diversas adaptações fisiológicas à baixa disponibilidade de fósforo (Pi) do solo. Este trabalho discute os custos fisiológicos e energéticos associados com essas estratégias, focado nas respostas da cana-de-açúcar (Saccharum spp.) à disponibilidade de Pi durante a simbiose com micorrizas arbusculares (Glomus clarum). Esses custos são importantes componentes para a adaptação a solos com baixo Pi, afetando a aquisição e conteúdo de fósforo; o crescimento e concentração de açúcares em tecidos vegetais. Plantas de cana-de-açúcar foram cultivadas em vasos com ou sem micorrizas (Glomus clarum), e sob a disponibilidade de baixo (20 mg kg-1) ou alto (202 mg kg-1) fósforo. Raízes e parte-aérea foram coletadas para as análises após 14, 30, 44 e 58 dias pós-inoculação (dpi) com Glomus clarum . A condição de BP causou a deficiência de Pi nas plantas, micorrízicas ou não. As plantas sob AP continham um teor foliar de Pi adequado, e partir dos 44 dpi acumularam pelo menos 6 vezes mais Pi parte-aérea, do que as cultivadas sob BP, efeito mais evidente nas micorrízicas. A eficiência de absorção, indicada pelo acúmulo de Pi na parte-aérea a uma dada biomassa da raiz, foi igual para todos os tratamentos, sugerindo que as eficiências radicular e micorrízica da absorção de Pi foram similares, independentemente da doses de Pi. A disponibilidade de fósforo não afetou a biomassa total das plantas, sendo as cultivadas sob BP mais eficientes na utilização deste nutriente. Por outro lado, as plantas micorrízicas suplementadas com BP apresentaram maior crescimento da raiz e redução na parte-aérea, resultando no aumento da proporção raiz:parte-aérea. Aos 58 dpi, a glicose, frutose e sacarose presente nas folhas de plantas micorrízicas foi 3,8, 2,3 e 2,4 vezes respectivamente mais concentrada do que nas não micorrízicas. Esses resultados sugerem que, nestas condições experimentais, o estabelecimento da simbiose não foi uma associação mutualística típica, afetando o perfil de crescimento e a alometria da cana cultivada com BP. As concentrações de fotoassimilados na folhas de planta micorrízicas indicam que houve aumentos nas taxas fotossintéticas, mas isso não resultou no maior crescimento do macrosimbionte. A tecnologia de amplificação quantitativa de transcritos reversos (RT-PCR) se tornou uma opção para a validação funcional de genes, com alta sensibilidade, acurada quantificação e eficácia. A quantificação relativa da expressão gênica é conseqüentemente fácil e determina a expressão de um gene em relação a outro expresso e relativamente constante. Neste trabalho foi analisada a variabilidade de expressão dos genes de cana-de-açúcar codificando a actina (Actina), gliceraldeido fosfato desidrogenase (GAPDH), tubulina (Tubulina), e ubiquitinas (UbiQ1 e UbiQ2) em diversos tecidos, e comparou-se a variabilidade obtida utilizando os programas Genorm e NormFinder. Em seguida, foram realizadas análises de expressão gênica utilizando o programa REST para a validação estatística da expressão de genes de cana-de-açúcar. O gene UbiQ1 foi mais estável nos tecidos ou órgãos testados: meristema, inflorescência, folha, colmo e raízes tratadas com alto e baixo fósforo. Tendo o gene UbiQ1 como referência, a expressão relativa dos genes transportadores de fosfato de alta afinidade de cana-de-açúcar PT7 e PT8 foi avaliada em amostras de raiz fertilizadas com alto ou baixo Pi, inoculadas ou não com fungo micorrízico e coletadas aos 58 dpi. Esses dois genes pertencem à família Pht1 de transportadores de fosfato e são similares aos ortólogos de arroz ORYsa;tPht1;7 e ORYsa;Pht1;8. Sob a deficiência de Pi, o transportador de fosfato PT7 foi induzido em raízes não colonizadas; já nas micorrízicas foi pouco expresso, sendo que altas taxas de colonização radicular suprimiram a expressão do PT7. O gene PT8 pouco variou sua expressão, sendo sutilmente mais expresso em plantas micorrízicas do que nas não micorrízicas sob o suprimento de BP. Estes resultados indicam que o PT7 é induzido em raízes sob estresse por fósforo e provavelmente associado à absorção radicular de Pi. Enquanto o gene PT8 possui uma modulação pouco variável provavelmente envolvido na manutenção do fluxo ou homeostase de Pi, possivelmente associado com a absorção radicular e micorrízica de fosfato. O PT7 e o PT8 foram expressos em tratamentos de médio/longo prazo, apresentando expressão ou indução em resposta a privação por Pi, o que é consistente com a função proposta de aquisição e mobilização de Pi para esta família de transportadores. A cana-de-açúcar micorrízica mostrou alta plasticidade de resposta ao BP / Plants display a wide array of physiological adaptations to low soil phosphorus (Pi) availability. This work discussed physiological and energetic costs associated with these strategies, focusing on sugarcane responses to Pi availability during the development of arbuscular mycorrhizal (AM) symbiosis. Such costs are important components of adaptation to low phosphorus soils affecting phosphorus acquisition and leve, growth and soluble sugars concentration in plant tissues. Sugarcane plants were grown in pots, with or without AM (Glomus clarum), and with low (20 mg kg-1) or high (200 mg kg-1) phosphorus supply. Roots and shoots were harvest for analysis after 14, 30, 44 and 58 days post-inoculation (dpi) with the fungus Glomus clarum,. The low Pi supply caused Pi deficiency in mycorrhizedl or non-mycorrhizedl plants. The efficiency of Pi absorption, indicated by shoot Pi accumulation in correlation to root biomass, suggested that root and mycorrhizal Pi absorption were similar, regardless of the Pi doses. Phosphorus availability did not affect the whole-plant biomass, and plants under low Pi supply, used more efficiently this nutrient. On the other hand, mycorrhized plants supplemented with low Pi presented the highest root growth and shoot reduction, resulting in high root:shoot ratio. At 58 dpi, glucose, fructose and sucrose concentrations in leaves of mycorrhized plants were 3.8, 2.3 and 2.4-fold higher respectively, than in non-mycorrhizedl plants. These results suggested that, under these experimental conditions, mycorrhizal symbiosis establishment was not a typical mutualistic association affecting sugarcane growth profile and allometry, when cultivated under low Pi. The photosynthate levels of leaves from mycorrhizd plants indicated an increase in photossynthetic rate but withut resulting in higher macrobiont growth. The quantitative amplification of reversed transcripts (RT-PCR) technology has become a method of choice for functional gene validation, with sensitiveness, accurate quantification and high-throughput. The relative quantification is easier determined by relative expression in comparison to a constitutively expressed refernce gene. Here, the expression variability of a sugarcane actin gene (Actin) glyceraldehydo phosphate dehydrogenase (GAPDH), tubulin (Tubulina), and ubiquitins (UbiQ1 and UbiQ2) from various tissues were analysed and compared based on the ranking list from the Genorm and NormFinder softwares. Expression analysis based on the REST software gave the proper statistic validation. UbiQ1 was the most stable gene among the candidate gen-references along the various tissues or organs tested: meristem, inflorescence, leaf, stem and roots treated with high and low phosphorus. Considering UbiQ1 as the reference gene, the relative expression of the sugarcane high-affinity phosphate transporters genes PT7 and PT8 were assessed from roots fertilized with low Pi or high Pi , inoculated or not with the mycorrhizal fungus, harvest 58 dpi. Both genes belong to the Pht1 Pi transporter and share similarity with the rice orthologs ORYsat;Pht1;7 and ORYsat;Pht1;8. Under Pi deficiency, the phosphate transporter PT7 was induced in non-colonized roots, but less expressed in mycorrhizedl ones, with high root colonization rate suppressing PT7 expression. PT8 showed low variability in expression, slightly more expressed in mycorrhized plants than in non-mycorhized plants under low Pi supply. These results indicated that PT7 was induced in Pi stressed roots, and possibly associated with the root Pi uptake, while PT8 had limited modulation in expression and probably involved on Pi fluxes or homeostasis, likely associated with both root and mycorrhizal phosphate uptake pathways. PT7 and PT8 were induced during medium/long-term treatments, showing induction or constant expression in both acquisition and mobilization of Pi in response to Pi deprivation, which is consistent with the proposed role of this tranporter family. Mycorrhizedl sugarcane showed a highly plastic response to low Pi

Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

La Tara (Caesalpinia spinosa) est une espèce forestière d’une grande importance en raison d’une forte demande sur le marché international pour les tanins présents dans ses gousses, et pour les gommes provenant de ses graines. Malgré son importance économique pour le Pérou, la majeure partie de la production provient de forêts naturelles non aménagées. Ces forêts présentent des problèmes de sol (érosion, faible fertilité, présence d’agents pathogènes, manque d'irrigation), qui conduisent à des rendements faibles. C’est pourquoi dans le présent travail, nous étudions les composantes microbiologiques du sol associé à cette culture, telles que les mycorhizes et les bactéries dont l’utilisation, selon de nombreuses études, s’est révélée être une alternative à l'utilisation d'engrais chimiques. Pour cela, nous avons procédé à l'analyse moléculaire de la diversité des champignons mycorhiziens arbusculaires par la technique de Miseq Illumina, ce qui nous a permis de mettre en évidence une prépondérance de Gloméracées parmi lesquelles les Rhizophagus spp. étaient retrouvés dans 70% des séquences. En outre, la dépendance de la Tara vis-à-vis de la mycorhization a été démontrée car, après avoir testé la mycorhization contrôlée de la Tara par Rhizophagus irregularis, il a été constaté que la croissance de Caesalpinia spinosa était considérablement améliorée, ainsi que l'absorption d'éléments nutritifs tels que l'azote (N) et le phosphore (P). Pour vérifier la capacité à noduler de la Tara, différents milieux de culture ont été utilisés ainsi que différentes conditions de croissance, en serre et in vitro. Ces expérimentations ont toutes montré que les racines de Tara ne présentaient pas de nodules, confirmant que cette légumineuse de la sous-famille des Caesalpinioideae est non nodulante. Par conséquent et afin d’étudier la diversité des rhizobia présents dans le sol de la plantation de Tara, nous avons utilisé en serre une plante-piège, le pois (Pisum sativum) car c’est une légumineuse nodulante et de plus est traditionnellement associée à la culture de Tara. Les rhizobia identifiés moléculairement se sont révélés très spécifiques et différents des rhizobia présents dans les sols extérieurs à la plantation de Tara. Plus particulièrement, ces rhizobia se sont révélés être phylogénétiquement proches de R. etli, R. phaseoli, R. pisi et R. leguminosarum. Enfin, un test d'inoculation contrôlée (in vitro) a été réalisé sur des plantules de pois, avec ces bactéries préalablement piégées et isolées du pois. Il a été observé que les rhizobia piégés à partir des sols collectés entre deux lignes de Tara et sur la ligne de plantation de Tara, ont stimulé la croissance du pois par rapport aux rhizobia présents dans les sols collectés à l'extérieur de la plantation. / The Tara (Caesalpinia spinosa) is a forest species of great importance due to its high demand in the international market for the tannins present in its pods and its seeds’ gum. Despite its great importance for Peru, most of the production comes from unmanaged natural forests. These forests present soil problems (e.g., erosion, low fertility, pathogens, lack of irrigation), which cause low yields. Therefore, in the present work we seek to study the soil components associated with Tara plantation , such as mycorrhizae and bacteria that have proved to be an alternative for reducing the use of chemical fertilizers in similar context (Aboubacar et al., Flores Chavez 20015, E and Saif 1987, Dia et al. 2010; Bilgo et al., 2013) . We used molecular analysis of the arbuscular diversity by the Miseq Illumina technique that allowed to verify the arbuscular diversity with a preponderance of Glomeraceae among which the Rhizophagus spp were found to be present in 70% of the sequences. In addition, the dependence of the Tara on obligatory mycorrhization was demonstrated, after testing the controlled mycorrhization of the Tara by the Rhizophagus irregularis. We found that the growth of this crop was significantly improved, as well as the absorption of nutrients such as nitrogen (N) and phosphorus (P).To check the nodulation of the Tara, different culture media were used (JenSen, sand mixture with Tara plantation soil, attapulgite mixture with Tara plantation soils) in greenhouse and in vitro condition. We did not manage to find rhizobial nodules in the roots which let us think that Tara is a non-nodular legume. Therefore, we used Pisum sativum as a trap plant to study the diversity of rhizobia present in the soil of the Tara plantation since this legume is often associated with Tara crop. The rhizobia found in the trap plant were very specific and different from the rhizobia present in soils outside the Tara plantation. Likewise, these rhizobia found to be phylogenetically close to R. etli, R. phaseoli, R. pisi and R. leguminosarum. Finally, we inoculated the trapped bacterias (in vitro) in Pisum sativum with the bacterias previously trapped and isolated from the pea (which grew in the green house); where it was observed that the rhizospheric bacteria of the zones IL (soil collected between two lines) and L (soil collected from the same line) from the plantation of Tara stimulated the growth of this crop with respect to the bacteria present in soils collected outside of the plantation (OP zone).