This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-07-05T12:16:24Z No. of bitstreams: 1 Emmanuella Vila Nova da Silva.pdf: 1939037 bytes, checksum: f521d3bf721007766ec42ccf2f7454a6 (MD5) / Made available in DSpace on 2016-07-05T12:16:24Z (GMT). No. of bitstreams: 1 Emmanuella Vila Nova da Silva.pdf: 1939037 bytes, checksum: f521d3bf721007766ec42ccf2f7454a6 (MD5) Previous issue date: 2012-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of plants symbiotically associated with N2 fixing bacteria and mycorrhizal fungi (AMF) provides an efficient strategy to accelerate the recovery of impacted areas and reduces its costs considerably. The term "mycorrhiza helper bacteria” (MHB) has been introduced and discussed due to the synergistic effect that this dual combination promotes to plants. They are bacteria associated with roots and AMF that selectively promote the establishment of symbiosis with fungi. Thus, the objectives were to verify the AMF activity in the area with native vegetation in the Pernambucano semiarid, municipality of Sertânia; determine glomerospores number and the most probable number (MPN) of infective propagules; quantify the content of glomalin-related protein in the soil and determine the feasibility of bacteria (MHB) co-inoculation and AMF mixture in “sabiá” (Mimosa caesalpiniifolia Benth) aiming at obtaining combinations and compatibility of symbiotic pairs, as well as to evaluate the mycorrhizal efficiency and colonization. The experiments were conducted in greenhouse of the Agronomic Institute of Pernambuco (IPA). 10 composite soil samples were collected with points were defined at random. Samples were homogenized and analyzed for physical and chemical characteristics. Composite samples were used for direct count (DC) and propagation of AMF for indirect count (IC) of spores, using trap- cultures and sorghum (Sorghum bicolor L. Moench) and peanut (Arachis hypogea L.) as host plants (experiment I). To determine the MPN of infective propagules of AMF in the Haplic Luvisol was used a system of serial dilution: 0, 1:10, 1:100 and 1:1000 with five replicates each, with maize (Zea mays L.) as host plant (experiment II). In the experiment III were used pots with Haplic Luvisol soil (8 kg pot-1) at pH 6.0 and the plant used was the “sabiá”. On seeding, inoculation with Burkholderia sabiae (BR 3405) and co-inoculation with BR3405 + MHB were performed and each seed was inoculated with 2 mL of specific medium for each of MHB bacteria and for the BR3405 containing 108 CFU mL-1. In the inoculation with AMF mixture was used 4 g pot-1 in the form of propagule containing approximately 670 spores. Plants were harvested at 110 days after planting (DAP) and the following variables were evaluated: shoot dry mass (SDM), root (RDM), RDM/SDM ratio, plant height (PH) on periods of 45, 90 and 110 days, root length (RL), total N accumulated in SDM (Nat), strains efficiency (E) and mycorrhizal colonization. The experimental design was randomized blocks, with 9 x 2 factorial arrangement plus an absolute control (AC) - without inoculation; MHB strains and one control treatment inoculated only with Burkholderia sabiae with and without AMF (AMF mixture) with 3 blocks . The experimental results show that the MPN of AMF infective propagules found in the city of Sertânia was 23 propagules cm-3. Soil proteins related to easily extractable glomalin (PSRGFE) and soil proteins related to total glomalin (PSRGT) were approximately 0.46 and 0.26 mg g soil-1, respectively. The AMF colonization combined with the bacteria was positive, as in the case of RL, treatments with BR 3405 + Azospirillum amazonenses (Y2) and BR 3405 + Herbaspirillum seropedicae (Z67) showed significant difference by the Tukey test (p <0.05) compared to the factor with and without AMF. Thereby ensuring that, in the presence of MHB bacteria there was increase in root length of “sabiá” plants. Strains efficiency showed better results when bacteria were in the presence of AMF and the treatment BR 3405 + Paenibacillus brasilensis (24) + AMF showed the best response. The treatments that received AMF were higher compared to the others on the variables SDM, RDM, E, Nac, coming to present on average 84% of root colonization. / A utilização de plantas associadas simbioticamente, com bactérias fixadoras de N2 e fungos micorrízicos arbusculares (FMA), constitui uma estratégia eficiente para acelerar a recuperação de áreas impactadas além de reduzir consideravelmente os custos com a mesma. O conceito de “mycorrhiza helper bacteria (MHB)” tem sido introduzido e discutido devido ao efeito sinergístico que essa dupla associação promove às plantas. São bactérias associadas com raízes e FMA que, seletivamente, promovem o estabelecimento da simbiose com os fungos. Deste modo, os objetivos deste trabalho foram verificar a atividade de FMA em área com vegetação nativa do semiárido Pernambucano, no município de Sertânia; determinar o número de glomerosporos e o número mais provável (NMP) de propágulos infectivos; quantificar o teor de proteínas do solo relacionadas à glomalina; determinar a viabilidade da co-inoculação entre bactérias (MHB) e mistura de FMA em sabiá (Mimosa caesalpiniifolia Benth) visando obter combinações e compatibilidade de pares simbióticos, assim como avaliar a eficiência e colonização micorrízica. Os experimentos foram conduzidos em casa de vegetação na Sede do Instituto Agronômico de Pernambuco (IPA). Foram coletadas 10 amostras compostas de solo, sendo os pontos definidos aleatoriamente. As amostras foram homogeneizadas e analisadas quanto às características físicas e químicas. Amostras compostas foram utilizadas para contagem direta (CD) e multiplicação de FMA para contagem indireta (CI) de esporos, com o uso de culturas-armadilha, empregando sorgo granífero (Sorghum bicolor L. Moench) e amendoim (Arachis hypogea L.) como plantas hospedeiras (experimento I). Para a determinação do NMP de propágulos infectivos de FMA no Luvissolo Háplico foi utilizado um sistema de diluição em série: 0, 1:10, 1:100 e 1:1000, com 5 repetições cada e, tendo o milho (Zea mays L.) como planta hospedeira (experimento II). No experimento III foram utilizados vasos com o solo Luvissolo Háplico (8 kg vaso-1) com pH 6,0 e a planta utilizada foi a sabiá. Na semeadura, foi efetuada inoculação com Burkholderia sabiae (BR 3405) e co-inoculação com BR3405 + MHB contendo 108 UFC mL-1. Na inoculação com a mistura do FMA, foram utilizados 4 g vaso-1 em forma de propágulo, contendo aproximadamente 670 esporos. A colheita foi realizada 110 dias após plantio (DAP) e foram avaliadas as seguintes variáveis: massa seca da parte aérea (MSPA), raiz (MSR), relação MSR/MSPA, altura de planta (AP) nos períodos de 45, 90 e 110 dias, comprimento da raiz (CR), N total acumulado na MSPA (Nac), eficiência das estirpes (E%) e colonização micorrízica. O delineamento experimental adotado foi em blocos casualizados, com arranjo fatorial 9 x 2 mais uma testemunha absoluta (TA) – sem inoculação; estirpes de MHB e um tratamento controle inoculado apenas com Burkholderia sabiae com e sem FMA (mistura de FMA) com 3 blocos. Os resultados dos experimentos mostram que o NMP de propágulos infectivos de FMA encontrados no município de Sertânia foi de 23 propágulos cm-3. As proteínas do solo relacionadas à glomalina facilmente extraível (PSRGFE) e as proteínas do solo relacionadas à glomalina total (PSRGT) ficaram em torno de 0,46 e 0,26 mg g solo-1, respectivamente. A colonização dos FMA em conjunto com as bactérias foi positiva, como no caso do CR, os tratamentos com BR 3405 + Azospirillum amazonenses (Y2) e BR 3405 + Herbaspirillum seropedicae (Z67) apresentaram diferença significativa pelo teste de Tukey (p<0,05) em relação ao fator com e sem FMA. Confirmando deste modo que, na presença das bactérias MHB houve aumento no comprimento do sistema radicular das plantas de sabiá. A eficiência das estirpes obteve os melhores resultados quando as bactérias estavam em presença de FMA e o tratamento BR 3405 + Paenibacillus brasilensis (24) + FMA foi o que obteve melhor resposta. Os tratamentos que receberam os FMA foram superiores em relação aos demais nas variáveis MSPA, MSR, E, Nac, chegando a apresentar uma média em torno de 84% de colonização radicular.

L'objectif de cette thèse est de contribuer à faire progresser les connaissances sur les interactions bénéfiques entre les plantes et les microorganismes en évaluant la contribution des systèmes de sécrétion de type III (SST3). Une synthèse des connaissances disponibles relatives aux SST3 chez les Pseudomonas non pathogènes, saprotrophes ou mutualistes, présentée chapitre I, montre que les SST3 ne sont pas cantonnés aux interactions parasites ou pathogènes avec les plantes. Dans l’étude expérimentale présentée chapitre II, nous avons utilisé différents génotypes de Medicago truncatula Gaertn. cv. Jemalong capables (Myc+) ou non (Myc-) d’établir une symbiose mycorhizienne. Ce travail nous a permis de montrer que les Pseudomonas spp. fluorescents possédant un SST3 (SST3+) sont préférentiellement associés aux racines mycorhizées des génotypes Myc+ de M. truncatula (J5 et TRV48) plutôt qu’aux racines du mutant Myc- (TRV25) et au sol nu. Ainsi, la plante seule n’est pas à l’origine de la présence accrue des Pseudomonas SST3+. La colonisation de la racine par les champignons mycorhizogènes à arbuscules (CMA), le développement du mycélium intraradiculaire et/ou la formation associée d’arbuscules, sont également déterminants. Dans l’étude présentée chapitre III, nous avons comparé les effets de la souche modèle promotrice de mycorhization (MHB) P. fluorescens C7R12 (SST3+) et de son mutant C7SM7 (SST3-), sur la mycorhization et la croissance de M. truncatula dans un sol non stérile. Ce travail a permis de montrer que le SST3 de C7R12 contribue à l’effet MHB de la bactérie. La promotion de la colonisation de la racine de M. truncatula par les CMA indigènes induite par le SST3 de C7R12 s’est traduite par une amélioration de la croissance de la plante. En revanche, l’inactivation du SST3 chez C7SM7 a eu un impact délétère sur la colonisation de la racine de M. truncatula par les CMA du sol étudié et sur la croissance de la plante. L’observation d’effets quantitatifs opposés entre C7R12 et C7SM7, nous a conduits à nous interroger sur l’existence d’un effet différentiel de l’inoculation de ces bactéries sur la structure et la diversité des communautés des microorganismes associés. Dans une étude présentée chapitre IV, le suivi dynamique en parallèle de la structure des communautés totales bactériennes (B-RISA) et fongiques (F-RISA) et de la colonisation de la racine par les CMA a été réalisée. Aucun effet de l’inoculation n’a été observé sur la structure des communautés fongiques de la rhizosphère ou des racines. En revanche, la structure des communautés bactériennes a varié selon que les plantes aient été inoculées ou non et selon la souche inoculée. Néanmoins, ces différences ont été observées plusieurs semaines après les effets de l’inoculation de C7R12 ou de C7SM7 sur la colonisation de la racine par les CMA. Ce décalage dans le temps, suggère que les différences observées dans la structure des communautés bactériennes pourraient être une conséquence plutôt qu’une cause des variations observées sur la mycorhization de M. truncatula. Nos résultats n’ont pas permis de mettre en évidence d’effets de l’inoculation sur la diversité des populations des bactéries fixatrices d’azote présentes dans les nodosités de M. truncatula. L’analyse des séquences de la grande sous-unité de l’ADN ribosomique (LSU rDNA) amplifiées à partir d’ADN extrait des racines, a montré pour les plantes inoculées et non inoculées, que les populations de CMA étaient majoritairement apparentées à Glomus intraradices. Un groupe d’isolats spécifiquement associé aux racines inoculées avec C7R12 et apparenté à G. claroideum a été décrit. Le groupe spécifique pourrait être associé à l’amélioration de la mycorhization observée dans les racines inoculées avec C7R12. Néanmoins, compte tenu de sa faible représentation numérique (8%), il semble probable que l’inoculation de C7R12 ait aussi un effet quantitatif sur la colonisation de la racine de M. truncatula par les CMA. etc / No abstract

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.