Memorial apresentado à Faculdade de Ciências Agronômicas da Universidade Estadual Paulista "Júlio de Mesquita Filho", Campus de Botucatu, para obtenção do título de Livre-Docente, junto à Disciplina "Microbiologia Geral" / Resumo: Estudou-se o efeito da inoculação com fungo micorrízico arbuscular (FMA), Glomus etunicatum, Glomus clarum, ou Scutelospora heterogama, e da adição de fósforo solúvel (15, 30, 45, 60, 120 e 240 mg de P kg-1 de terra) sobre as variáveis altura, número de folhas, diâmetro do caule, massas vegetais aérea e radicular, teores de nutrientes nas folhas e colonização micorrízica no porta-enxerto limoeiro "Cravo" (Citrus limonia (L.) Osbeck). Os FMAs, Glomus etunicatum e Glomus clarum, e doses crescentes de fósforo exerceram efeitos significativos sobre essas variáveis. O efeito da inoculação sobre as variáveis de crescimento vegetal foi mais acentuado nos tratamentos com adição de 30 a 60 mg de P kg-1 de terra. Para o teor de P foliar, o efeito da inoculação foi mais acentuado nos tratamentos adicionados de 120 a 240 mg de P kg-1 de terra. A inoculação com estes FMAs aumentou a eficiência do limoeiro "Cravo" em absorver nutrientes, tanto que as variáveis estudadas em plantas inoculadas na ausência de adubação fosfática superaram às de plantas não inoculadas em substrato adicionado de 240 mg de P kg-1 de terra. / Abstract: The effects of arbuscular mycorrhizal fungus (AMF) Glomus etunicatum, Glomus clarum or Scutelospora heterogama inoculation and soluble phosphate addition (15, 30, 45, 60, 120 and 240 mg P kg-£ of soil) on the height, leaf number, stem diameter, shoot and root mass, nutrients in foliar mass and mycorrhizal colonization parameters on Rangpur lime rootstocks plants were studied. AMF inoculation and soluble phosphate levels increased these parameters. Inoculation effect on the plant growth parameters was more intense at 30 to 60 mg P kg-1 soil. AMF inoculation effect on the leaf P was more intense at 120 to 240 mg P kg-1 soil addition. AMF inoculation increased citrus efficiency to absorb nutrients so that the parameters studied of inoculated plants without phosphate addition did not differ of non-inoculated plants in substract with 240 mg P kg-1 soil addition.

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

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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The production expansion areas of soybean monoculture system result in increased fertilizers and pesticides application, and the intensification of some phytosanitary problems such as the damage caused by plant-parasitic nematodes. The arbuscular mycorrhizal fungi (AMF) increase plant growth and reduce the problems caused by nematodes in soybean. The objective of this work was to study the effect of arbuscular mycorrhizal fungi Rhizophagus clarus in the growth of soybean plants and the nematode Pratylenchus brachyurus suppression. Two trials were conducted in a greenhouse. In the first experiment evaluated the growth of soybeans in the presence and absence of the mycorrhizal fungus R. clarus. In the second trial was carry out the damage caused by the P. brachyurus in soybean grown in the presence and absence of R. clarus. In soybean flowering period evaluated the height, stem diameter, the dry matter of root and shoot, nodulation, the relative chlorophyll index, the nutrient content of the plant tissue, the number of the nematode in soil and penetrated to the root system, the reproduction factor of the nematode, the number of fungal spores in the soil and mycorrhizal colonization of roots. The results showed that mycorrhiza increased the growth, nodulation and nutrient uptake of soybean plants grown in soil with or without nematode. The mycorrhizal colonization reduced the penetration of the nematode in soybean roots, despite the greater number of nematodes in the soil. The presence of the nematode influenced mycorrhizal colonization of soybean plants. The use of sustainable management practices that favor the populations of AMF in the soil, can be a strategy for increasing and/or maintaining crop yields and reducing the affect caused by the P. brachyurus. / A expansão das áreas de produção e o sistema de monocultivo da soja resultam no aumento da aplicação de fertilizantes e agrotóxicos, e na intensificação de alguns problemas fitossanitários, como os danos causados pelos fitonematoides. Os fungos micorrízicos arbusculares podem contribuir para aumentar o crescimento das plantas e reduzir os problemas causados pelos nematoides na cultura da soja. O objetivo do presente trabalho foi estudar o efeito do fungo micorrízico arbuscular Rhizophagus clarus no crescimento das plantas de soja e na supressão do nematoide Pratylenchus brachyurus. Dois ensaios foram conduzidos em casa de vegetação. No primeiro ensaio foi avaliado o crescimento da soja na presença e na ausência do fungo micorrízico R. clarus. No segundo ensaio foi avaliado os danos causados pelo nematoide P. brachyurus na cultura da soja cultivada na presença e na ausência do R. clarus. No período de florescimento da soja avaliou-se a altura, o diâmetro de colmo, a fitomassa da parte aérea e radicular, a nodulação, o índice relativo de clorofila, o teor de nutrientes do tecido vegetal, o número de P. brachyurus no solo e penetrados no sistema radicular, o fator de reprodução dos nematoides, o número de esporos fúngicos no solo e a colonização micorrízica das raízes. Os resultados demonstraram que a micorrização aumentou o crescimento, a nodulação e a absorção de nutrientes das plantas de soja, cultivadas em solo com ou sem o nematoide. A colonização micorrízica reduziu a penetração do P. brachyurus nas raízes da soja, apesar do maior número de nematoides no solo. A presença do nematoide influenciou a colonização micorrízica das plantas de soja. A adoção de práticas de manejo sustentáveis que favoreçam as populações de FMAs no solo, pode ser uma estratégia para o aumento e/ou manutenção da produtividade das lavouras e redução dos danos causado pelo P. brachyurus.

Les activités industrielles, la production d’énergie le transport et l’urbanisation ont engendré de sérieux problèmes environnementaux qui ont des effets néfastes non seulement pour les divers écosystèmes, mais aussi pour la santé des Humains. Il existe plusieurs méthodes de réhabilitation des sites contaminés. Les méthodes dites conventionnelles consistent le plus souvent à excaver, transporter et entreposer des sols dans des sites d’enfouissements, alors que d’autres technologies utilisent des traitements physico-chimiques ou l’incinération des polluants. Les inconvénients majeur de ces méthodes en sont le coût élevé, l’émission des gaz à effet de serre et la destruction des habitats. Cependant, plusieurs technologies ont émergé ces dernières décennies. Parmi ces technologies émergentes, la phytoremédiation est une méthode prometteuse et dont l’efficacité devienne de plus en plus reconnue. La phytoremédiation consiste à utiliser des plantes et les microbes qui leurs sont associés pour dégrader, extraire ou stabiliser les polluants du sol aussi bien organiques qu’inorganiques. Parmi les microbes associés aux racines des plantes, on trouve les champignons mycorhiziens arbusculaires (CMA) dont le rôle en phytoremédiation a été montré. Cependant, la diversité et les changements des structures des communautés de ces champignons dans des sites hautement contaminés et en association avec les populations des plantes qui poussent spontanément dans ces sites demeurent méconnues. L’objectif de mon projet de maitrise consiste à étudier la diversité et la structure des communautés des CMA dans les racines et les sols rhizosphériques de trois espèces de plantes Eleocharis elliptica, Populus tremuloides et Persicaria maculosa qui poussent spontanément dans des bassins d’une ancienne raffinerie pétro-chimique. J’ai échantillonné trois individus par espèce de plante dans trois bassins qui ont montré des concentrations différentes des polluants pétroliers. J’ai utilisé l’approche de la PCR conventionnelle, le clonage et le séquençage en ciblant le gène 18S de l’ARN ribosomique autant sur des échantillons de racines et des que sur ceux de sols rhizophériques. J’ai analysé au minimum 48 clones par échantillon. L’analyse de la diversité Beta a montré que la structure des communautés des CMA était significativement différente selon les biotopes (racines et sols rhizosphèriques) et les concentrations de contaminants pétroliers. Mes résultats ont montré que l'identité de la plante et la concentration de contaminants ont fortement influencé la structure des communautés de CMA. J’ai aussi observé qu’en plus de l’effet des facteurs biotiques et abiotiques mentionnés ci-dessus, plusieurs OTUs de CMA sont corrélés soit positivement ou négativement entre eux et aussi avec différents types de polluants d'hydrocarbures pétroliers. Cette étude a permis de comprendre les facteurs qui influencent les changements des structures des communautés des CMA et pourrait nous aider à améliorer l’efficacité de la phytoremédiation avec des plantes indigènes poussant spontanément sur des sites hautement contaminés par des hydrocarbures pétroliers. / Industrial activities, energy production, transportation, and urbanization have led to serious environmental problems that have negative effects not only for the natural ecosystems, but also for the human health. Several methods of rehabilitation of contaminated sites such as conventional methods consisting on excavation, transportation and storage of contaminated soils in landfills (known as Dig and Dump), as well as other technologies that use physical and chemical treatments or incineration of polluted soil pollutants, have been largely utilized. However, these methods are very costly and not environmental-friendly because of greenhouse gas emission and destruction of habitats. Several green technologies have emerged in recent decades. Among these emerging technologies, phytoremediation is a promising method whose effectiveness becomes increasingly recognized worldwide. Phytoremediation uses plant and their associated microbes to degrade, uptake or sequestrate organic and inorganic pollutants. Arbuscular mycorrhizal fungi (AMF) are among microbes that live intimately with plant root where they form a symbiosis known as arbuscular mycorrhiza. The objective of my master project was to study the diversity and changes of community structure of AMF in roots and rhizospheric soils of three native plant species Eleocharis elliptica, Populus tremuloides and Persicaria maculosa growing in petroleum-contaminated sedimentation basins of a former petro-chemical plant. I used conventional PCR, cloning and sequencing approach targeting 18S rRNA gene to investigate AMF community structure. I analyzed at minimum 48 clones for each sample. Beta diversity analyses showed that AMF community structure was significantly different across biotopes (roots and rhizospheric soils) and different concentrations of petroleum hydrocarbon contamination. Our results showed that plant identity and concentrations of petroleum hydrocarbon contaminations strongly influenced the AMF community structure as well as the inter-specific relationship among AMF taxa. Moreover, with consideration of both biotic and abiotic factors, we found that several AMF OTUs showed positive and negative correlations between each other and also with petroleum hydrocarbon pollutants. My study brings us in-valuable information to apply AMF for the phytoremediation in the future.

La symbiose mycorhizienne à arbuscule est une interaction bénéfique entre les champignons du phylum Glomeromycota et près de 80% des espèces de plantes terrestres. Elle est caractérisée par un échange réciproque de nutriments dans lequel le champignon fournit des sels minéraux à la plante en échange de sucres issus de la photosynthèse. Cependant, cette "alimentation" du champignon au cours de la symbiose représente un coût carbone important pour la plante. Ainsi, les plantes doivent strictement maitriser le développement des champignons symbiotiques dans les racines. Ce contrôle est appelé autorégulation. Plusieurs protéines ont été démontrées comme étant importantes pour la régulation des différentes étapes de la colonisation : la stimulation de la croissance fongique dans la rhizosphère par les strigolactones, l'entrée dans les racines, la prolifération des hyphes au sein des racines et la formation des arbuscules. Dans ce travail, nous avons examiné plus en détail le rôle de deux de ces protéines connues pour être impliquées dans le processus de mycorhization, les facteurs de transcription NSP1 et NSP2 (Nodulation Signaling Pathway). Nous avons d'abord pu confirmer dans les racines de M. truncatula en conditions non-symbiotiques, l'implication directe de NSP1 dans la régulation de deux gènes de biosynthèse des strigolactones, DWARF27 (D27) et MORE AXILLARY GROWTH (MAX1). Ensuite, nous avons montré que NSP1, contrairement à NSP2, favorise l'entrée du champignon dans la racine, sans doute due à l'induction de la synthèse des strigolactones stimulant le champignon, via l'activation de D27 et de MAX1. Ensuite, au cours des étapes ultérieures de la mycorhization, nous avons montré que dans les tissus colonisés, NSP1 est absent et que l'induction de D27 et de MAX1 n'était plus NSP1 dépendante. À cette étape, l'expression de la protéine NSP1 est localisée dans les cellules justes en amont du front de colonisation fongique. Là, elle contrôle négativement la propagation des hyphes dans la racine et positivement la formation des arbuscules. En revanche, NSP2 est présente dans le tissu colonisé où elle favorise la propagation des hyphes et le développement des arbuscules, peut-être en interaction avec d'autres facteurs. Nous avons également montré chez M. truncatula que si les protéines NSP1 sont absentes des tissus colonisés, les transcrits de NSP1 sont présents. De façon inattendue, nous avons mis en évidence que l'ARN messager de NSP1 avait la capacité de protéger l'ARN messager de NSP2 contre sa dégradation par le microARN (miR171h), par une action de piégeage du miR171h, appelé effet mimicry. Ceci est la première démonstration qu'une molécule d'ARN codante peut être la cible mimétique d'un microARN. Dans notre contexte d'étude cette constatation révèle que les transcrits de NSP1 permettent une régulation positive de l'expression de NSP2, et met en lumière un niveau de complexité supplémentaire dans le rôle de ces deux facteurs de transcription dans la symbiose mycorhizienne. Enfin, dans la tomate, nous avons montré que Sl-NSP1 pourrait être directement ou indirectement régulée par une protéine AUX / IAA impliquée dans la réponse précoce à l'auxine, Sl-IAA27. Ce lien avec l'auxine nous fait présumer que cette AUX/AAI est un nouveau composant de la voie de signalisation du contrôle de la colonisation fongique dans la tomate, et nous proposons qu'il puisse avoir un rôle dans le contrôle de la biosynthèse des strigolactones via la régulation de Sl-NSP1. L'ensemble de ce travail fournit de nouvelles pièces du puzzle constituant la symbiose mycorhizienne et montre l'importance de l'analyse des régulations spatiotemporelles pour une meilleure compréhension de ces processus biologiques extrêmement complexes. / The arbuscular mycorrhiza (AM), a symbiosis between fungi from the phylum Glomeromycota and nearly 80% of terrestrial plant species. It is characterized by a two-way exchange in which the fungus provides mineral nutrients to the plant in exchange for carbohydrates. However this "feeding" of the fungus during the symbiotic process represents a significant carbon cost for the plant. To maintain a mutualistic interaction the two symbiotic partners have to strictly control the extent of fungal development in the roots. This control is called autoregulation. Several proteins have been found to be important for the regulation of the different mycorrhizal steps: the stimulation of fungal growth in the rhizosphere by the strigolactones, the fungal entrance in the roots, the hyphal proliferation in the roots and the arbuscule formation. In this work we examine in more detail the role of two of these proteins known to be involved in the mycorrhization process, the transcriptional factors NSP1 and NSP2 (Nodulation Signaling Pathway). We first confirm in M. truncatula roots the direct implication of NSP1 in the regulation of two strigolactone biosynthesis genes, DWARF27 (D27) and MAX1, during the asymbiotic conditions. Then, we show that NSP1, unlike NSP2, is a factor that promotes the fungal entries in the root, presumably due to its activation of D27 and MAX1 resulting in a stimulation of strigolactone synthesis and presymbiotic fungal growth. Next, during the later stages of mycorrhization, we highlight that in the colonized tissues NSP1 is absent and the induction of both D27 and MAX1 is not anymore NSP1 dependent. NSP1 protein is then localized in cells which are not yet colonized but are close to a colonization zone. There, it controls negatively the hyphal propagation in the root and positively the formation of arbuscules. In contrast, NSP2 is present in the colonized tissue where it promotes hyphal propagation and arbuscule development, perhaps by interacting with other proteins. We also show that if NSP1 proteins are absent of the colonized tissues, NSP1 transcripts are present. Unexpectedly, we unveil that in those colonized cells, NSP1 mRNA can protect, by a micro RNA (miR171h) decoy action called target mimicry, NSP2 mRNA against miR171h-mediated degradation. This is the first demonstration that a coding RNA molecule can be a target mimic for a microRNA. In our context this finding reveals a positive regulation of NSP2 expression by NSP1 transcripts and brings to light an additional layer of complexity in the mycorrhizal dual role of these two transcription factors. Finally, in tomato, we highlight that SlNSP1 could be directly or indirectly regulated by the AUX/IAA protein, SlIAA27. As a link with auxin we presume that this AUX/IAA protein is a new component of the signaling pathway controlling AM fungal colonization in tomato, and we propose that it controls strigolactone biosynthesis via the regulation of SlNSP1. Overall our work provides new pieces of the mycorrhizal puzzle and shows how important it is to perform spatiotemporal investigations for a better understanding of highly integrated and complex biological processes.

Dynamics of carbon and phosphorus flows in arbuscular mycorrhizal symbiosis Mgr. Tereza Konvalinková (doctoral thesis) Abstract Arbuscular mycorrhizal fungi (AMF) are widespread and highly specialized root symbionts, which gain all of their carbon (C) from the hosts, supplying plants with mineral nutrients (particularly with phosphorus, P) in return. This thesis focuses on the size and flexibility of C and P flows in arbuscular mycorrhiza in relation to environmental conditions, in particular to light and P availability. The indications that the symbiotic flows are regulated actively by both partners are discussed. The main findings are presented as a compilation of separate scientific works (two research articles, one review and one book section). A glasshouse experiment has shown that both mycorrhizal benefits and mycorrhizal colonization of medic (Medicago truncatula) by an AMF species (R. irregularis) decline along the gradient of decreasing light intensity. Interestingly, morphological adaptation of medic to the long-term light deprivation was boosted by mycorrhiza, probably because of C demand of AMF and due to the improved nutrition of the mycorrhizal plants. On the other hand, sudden 6-day shading caused rapid decline of shoot P content of mycorrhizal plants, accompanied with the accumulation of P...

The aim of this project is to investigate a cultural history of mushroom collecting in Sweden from the 19th century to the present with a focus on connections between aesthetics and the environment.  Collecting is defined broadly as gathering, storing, and accumulating.  This definition encompasses mycologists, mushroom enthusiasts, botanists, and natural historians documenting and preserving as many species as possible.  But it also includes collecting in the sense of leisurely mushroom collectors taking a part of the environment home with them to eat, store, or learn from. A history of mushroom collecting in Sweden is framed that does not only focus on edible mushrooms or scientific value, but emphasizes their linkages to place, memory, conservation, sociality, and embodied knowledge. Mushroom aesthetics are a starting point for exploring wider human connections to the environment and human perceptions of nature.   Collecting is presented as a process which is argued to be a means for constant dialogue with the environment. The cyclical collecting process is broken into stages that are discussed in designated chapters: Hunting, Identification and Assessment, and Storage and Sharing. Aesthetic aspects of mushroom collecting in Sweden are examined within these stages applying visual and discourse analyses to archival images, questionnaires, historic cookbook recipes, and mushroom identification books. Importance is also assigned to fully immersed aesthetic experiences and specific sensory stimuli that facilitate interconnection with non-human actors. Immersed aesthetic experiences are argued to be significant in their ability to democratize aesthetic appreciation of nature, in contrast to historical associations of aesthetics with taste and high culture. Fluctuating historical judgments are mapped about mushrooms, highlighting the framing of nature as a productive asset. Mushroom exhibitions are shown to be a point of collective meaning making, where aspects of natural time according to mushrooms challenge anthropocentric notions of temporality. This thesis through its focus on aesthetics in mushroom collecting reveals spaces of uncertainty and dynamic fluctuation in human-nature relationships, as well as a sense of value for being physically present and part of environments.

Les toits verts sont des écosystèmes d’une grande importance pour les milieux urbains. Cependant, le microbiome du sol est peu considéré dans l’aménagement de ces habitats, alors qu’il est pourtant à la base de nombreux services écosystémique, dont le cyclage des nutriments et la productivité primaire. Il est donc nécessaire de s’intéresser davantage à l’assemblage de ce microbiome, de manière à éventuellement mieux manipuler ces communautés pour favoriser le maintien des services écosystémiques. Nous avons donc échantillonné le sol 19 toits verts en plus de cinq grands parcs urbains afin d’étudier les communautés bactériennes, fongiques et mycorhiziennes de ces habitats. Contrairement à ce que prédisent les théories classiques en écologie, les communautés microbiennes des sols des toits verts sont abondantes et diversifiées même dans les toits les plus jeunes. De plus ces communautés ne sont pas dominées par des microorganismes reconnus comme étant tolérants au stress. Ainsi, les limites à la dispersion ne semblent pas affecter ces communautés microbiennes isolées. Une grande variation dans les structures des communautés est restée non expliquée, montrant peu d’évidences d’assemblages déterministes. Ce phénomène pourrait être dû à une plus grande importance de déterminants ou processus stochastiques. Nous avons aussi observé ce phénomène chez les champignons mycorhiziens avec une plus grande abondance des espèces fréquentes régionalement et globalement. Cela montre l’importance du pool régional d’espèces pour l’assemblage des communautés des toits verts. Les toits échantillonnés ont des microbiomes uniques aux parcs environnants, avec une faible abondance de certains groupes taxonomiques, comme les Thaumarchaeota (procaryotes nitrificateurs), les actinobactéries (saprotrophes), ou les Gigasporaceae (champignons mycorhiziens produisant un important réseau d’hyphes extraracinaires). Ces profils microbiens uniques pourraient induire des conséquences biogéochimiques importantes sur les processus écosystémiques du sol des toits verts. Les recherches futures devraient évaluer les liens entre la structure du microbiome et les fonctions écosystémiques rendus par les toits verts. / Green roofs are novel ecosystems of great importance for urban environments. However, green roof soil microbiome has received little attention, even though it supports numerous ecosystem services. It is therefore necessary to pay more attention to the green roof soil microbiome assembly and eventually better manipulate it to promote the maintenance of ecosystem services, as nutrient cycling and primary productivity. We sampled 19 green roofs in addition to five large urban parks to study the bacterial, fungal and mycorrhizal communities in these habitats. Contrary to what was expected under classic ecological theories, microbial communities were abundant and diverse, even on the youngest roofs. Moreover, green roofs soils were not dominated by microorganisms known to be particularly stress tolerant. Dispersal limitation did not appear to affect the green roof soil communities. High level of variation in community structure remained unexplained, showing little evidence of deterministic assembly. This phenomenom may be a sign of stochastic assembly in these habitats. It was partly observed for mycorrhizal fungi with greater abundance of regionally and globally frequent species. This suggests the importance of regional species pool for community assembly in green roofs. The sampled roofs showed unique microbiomes, with low abundance of some taxonomic groups, such as the Thaumarchaeota (nytrifying prokaryotes), Actinobacteria (sapotrophs), or Gigasporaceae (mycorrhizal fungi producing an important external hyphae network). This could have important biogeochemical consequences on green roofs. Much insight will be gained from future research looking at the links between microbiome composition and the ecosystem services provided by green roofs.

L’utilisation d’inoculants de champignon mycorhizien arbusculaire (CMA) et de bactéries promotrices de la croissance de la plante (PGPR) comme biofertilisants pour améliorer la croissance de la plante et augmenter les rendements des cultures connait de plus en plus un intérêt remarquable. Ces inoculants peuvent influencer les relations trophiques à travers les changements induits au niveau de la plante. Cependant, en contexte agroécologique, il est difficile de prédire les effets de leur application sur les niveaux trophiques supérieurs, notamment sur les insectes phytophages et leurs ennemis naturels. Ainsi, l’objectif de ce projet de thèse était d’évaluer les effets des inoculants de champignon mycorhizien et bactériens sur les insectes du soja. Spécifiquement il est question : 1) d’évaluer l’influence sur le puceron du soja Aphis glycines (2e niveau trophique), de la symbiose tripartite entre un CMA Rhizophagus irregularis, une bactérie rhizobium Bradyrhizobium japonicum et le soja Glycine max ; 2) de déterminer l’impact des inoculants de R. irregularis et de B. japonicum associés au soja sur les ennemis naturels du puceron de soja, la coccinelle Coleomegilla maculata et le parasitoïde Aphelinus certus (3e niveau trophique) ; 3) d’évaluer les effets de la co-inoculation des inoculants de R. irregularis, B. japonicum et de Bacillus pumilus sur les insectes phytophages et leurs ennemis naturels associés au soja au champ. Les expériences en chambre de croissance (objectifs 1 et 2) ont montré une altération des traits de performance des plantes en présence des inoculants comparées aux plantes témoins. Les différences de colonisation mycorhizienne et de nodulation, observées entre les traitements sont une preuve du fonctionnement de l’inoculation. Avec le double inoculant, j’ai observé une augmentation de la biomasse de la plante, des concentrations en azote et carbone, et une diminution de la concentration en phosphore. Avec le rhizobium seul, j’ai obtenu les mêmes effets, sauf pour la biomasse racinaire qui n’a pas été affectée. Avec le CMA seul, une augmentation de la concentration en phosphore a été observée, mais aucun des autres paramètres de la plante n'a été affecté. Au deuxième niveau trophique sur le puceron du soja, le soja inoculé avec le double inoculant CMA+rhizobium, suivi du rhizobium seul, ont augmenté significativement la densité de la population de pucerons, alors qu’aucun des inoculants n’a eu un effet sur le fitness du puceron. En revanche, aucun des traits de performance du puceron (fécondité et taille des individus) n’a été affecté par la présence de l’inoculant CMA seul. D’autre part, j’ai noté une corrélation positive entre la concentration en azote et le taux de reproduction des pucerons, alors qu’avec la concentration en phosphore, la corrélation avec la colonie des pucerons est négative. Quant au troisième niveau trophique, une réduction significative du taux de parasitisme chez A. certus a été observée en présence du rhizobium seul, mais aucun autre paramètre n’a été affecté comparativement aux autres traitements : contrôle, CMA et double inoculant (CMA+rhizobium). Avec le prédateur C. maculata, aucun paramètre mesuré n’a été affecté par les effets indirects des inoculants. Il ressort de l’évaluation que j’ai faite au champ (objectif 3), une augmentation de l'abondance des insectes piqueurs-suceurs avec le triple inoculant (CMA+rhizobium+Bacillus), mais aucune différence entre les traitements pour les autres groupes d'insectes (broyeurs, et ennemis naturels des pucerons). Aucun groupe fonctionnel n’a été affecté en présence du double inoculant CMA+rhizobium sauf le puceron du soja, A. glycines, qui a vu sa population décroître mais seulement dans les parcelles fertilisées en potassium. J’ai également montré que les abondances des insectes piqueurs-suceurs et des insectes broyeurs, et la diversité alpha des insectes phytophages étaient toutes corrélées négativement avec la colonisation mycorhizienne globale. Ces résultats confirment que la co-inoculation de deux symbiotes peut non seulement améliorer les performances des plantes mais aussi celles des insectes phytophages au-delà de ce que chaque symbiote peut apporter seul. Au moins dans notre système d'étude en chambre de croissance, il semble que les avantages que les symbioses microbe-plante confèrent au deuxième niveau trophique avec une augmentation de la population de pucerons sont peu transférés au troisième niveau sur les ennemis naturels du puceron. Dans un système agricole où les agriculteurs veulent bénéficier des retombées de ces inoculants, notamment dans la gestion des insectes associés aux cultures, les CMA semblent favorables s’ils sont utilisés seuls et du fait de leur potentiel à réduire les insectes ravageurs. / The use of arbuscular mycorrhizal fungi (AMF) and plant growth promoting bacteria (PGPR) inoculants, as biofertilizers to boost plant growth and increase crop yields, is gaining interest. These inoculants can also influence trophic relationships through changes on plant. However, in an agroecological context, it is difficult to predict the effects of their application on higher trophic levels, namely phytophagous insects, and their natural enemies. The objective of this thesis research is to evaluate the effects of mycorrhizal fungi and bacterial inoculants on soybean-associated insects. Specifically, it is to: 1) evaluate the influence on the soybean aphid, Aphis glycines (2nd trophic level), of the tripartite symbiosis between an AMF, Rhizophagus irregularis, a rhizobium bacterium, Bradyrhizobium japonicum, and the soybean, Glycine max; 2) determinate the impact of R. irregularis and B. japonicum inoculants associated with soybean on the natural enemies of soybean aphid, the ladybug Coleomegilla maculata and the parasitoid wasp Aphelinus certus (3rd trophic level); 3) evaluate the effects of co-inoculation of R. irregularis, B. japonicum and Bacillus pumilus inoculants on soybean-associated phytophagous insects and their natural enemies in an agricultural field conditions. The walk-in growth room experiments (objectives 1 and 2) showed an alteration of the performance traits of the plants in the presence of the inoculants compared to control plants. The differences in mycorrhizal colonization and nodulation, observed between treatments, are evidence that the inoculation works. With the double inoculant, I observed an increase in plant biomass, nitrogen and carbon concentrations, and a decrease in phosphorus concentration. With rhizobium inoculation alone, without AMF, I obtained the same effects, except the root biomass that was not affected. With AMF alone, an increase in phosphorus concentration was observed, but none of the other plant parameters were affected. At second trophic level, inoculation of soybeans with the dual inoculant AMF-rhizobium, followed by rhizobium alone, significantly increased aphid population density, while none of inoculant influenced the fitness of the aphid. However, AMF alone did not affect any parameter traits (reproduction and fitness) of the aphid. I noted a positive correlation between nitrogen concentration and aphid reproductive rate, whereas the correlation between phosphorus concentration and aphid colony growth was negative. At the third trophic level, a significant reduction in the rate of parasitism of A. certus was observed in the presence of rhizobium inoculant alone, but no other parameters were affected with any other treatments, namely the control, the AMF inoculant, or the double inoculant (AMF+rhizobium). With the predator C. maculata, no measured parameters were affected by the indirect effects of any of the inoculant treatments. During my field assessment (objective 3), I observed an increase in the abundance of piercing-sucking insects with the triple inoculant (AMF+rhizobium+Bacillus), but no differences between treatments for other insect groups (chewing insects, or natural enemies of aphids). No functional group was affected by the presence of the double inoculant, CMA+rhizobium except the soybean aphid, A. glycines, which saw its population decrease in potassium-fertilized plots. I also showed that the abundance of piercing-sucking insects, chewing insects, and the alpha diversity of phytophagous insects were all negatively correlated with mycorrhizal colonization. The results of my studies confirm that co-inoculation of two symbionts can not only improve plant performance but also that of phytophagous insects beyond what each symbiont can provide alone. At least in our growth chamber study system, it appears that the benefits that microbe-plant symbioses confer to the second trophic level, with an increase in aphid population, are little transferred to the third level on the natural enemies of the soybean aphid. In agricultural system where farmers seek the benefits of these inoculants, especially in the management of insects associated with crops, AMF seem favorable when they are used alone and because of their potential to reduce insect pests.