Made available in DSpace on 2017-07-25T19:31:00Z (GMT). No. of bitstreams: 1 Kamila Santana.pdf: 1363071 bytes, checksum: 4f5da813f22ced41d3b26851a927779f (MD5) Previous issue date: 2016-06-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The blueberry is an agricultural crops recently introduced in Brazil and lots of researches are turning to develop and adapt it to the country. Due to its low amount of root hair, it forms associations with microorganisms to expand its zone of nutrient uptake, being the nitrogen the most required element. At this work it was observed the interaction of this cultivation with the inoculum Azospirilum brasilense and Trichodema asperellum into different substrates or doses of nitrogen fertilization. The experiment was performed with cv. Climax and the plants conditioned in pots in greenhouse, under trial design of randomized blocks, factorial 3x3. The considered factors were inoculation (A. brasilense, T. asperellum or without inoculation) and formulation of the substrates (5; 20 e 35% of organic matter – hardened cattle manure) or, at the second experiment, inoculation (A. brasilense, T. asperellum or without inoculation) and doses of nitrogen (0, 10 and 15 g f N plant-1 – provided as urea). The larger quantity of organic matter provided the highest averages relating to physiological (transpiration, water use efficiency and carboxylation efficiency) and growth variables (foliar limb area, total foliar area and rate of relative growth), although, for some of them, it has not differed from plants grown with 20% of organic matter. With 30% of O.M., the inoculation with T. asperellum showed superior averages in CO2 assimilation (A), carboxylation efficiency (A/Ci) and rate of relative growth (RRG). The nitrogen doses positively affected A/Ci, clorophyll amounts, foliar area (limb and total) and foliar nitrogen concentration, although it not differing between 10 or 15g of N plant-1. For the accumulation of the nutrients Ca, Mg, S, Fe and Zn, the nitrogen fertilization did not represent higher foliar levels, even though this might be related to a dilution effect. / O mirtileiro é uma cultura recentemente introduzida no Brasil, sendo que várias pesquisas tem-se voltado para seu melhor desenvolvimento e adaptação no país. Por possuir poucos pelos radiculares, forma associações com microrganismos afim de aumentar sua zona de absorção de nutrientes, sendo o nitrogênio o elemento absorvido em maior quantidade. Neste trabalho, observou-se a interação dessa cultura com os inóculos Azospirillum brasilense e Trichoderma asperellum em diferentes substratos ou doses de adubação nitrogenada. O experimento foi realizado com a cv. Clímax e as plantas acondicionadas em vasos, em casa de vegetação, sob delineamento experimental de blocos casualizados em esquema fatorial 3x3. Os fatores analisados foram inoculação (A. brasilense, T. asperellum ou sem inoculação) e formulação dos substratos (5; 20 e 35% de matéria orgânica – esterco bovino curtido) ou, no segundo experimento, inoculação (A. brasilense, T. asperellum ou sem inoculação) e doses de nitrogênio (0, 10 e 15 g de N planta-1 – fornecido como ureia). A maior proporção de matéria orgânica propiciou as maiores médias em relação a variáveis fisiológicas (transpiração, eficiência no uso da água e eficiência de carboxilação) e de crescimento (área de limbo foliar, área foliar total e taxa de crescimento relativo), embora, para outras, não tenha diferido das plantas cultivadas com 20% de matéria orgânica. Com 35% de M.O., a inoculação com T. asperellum apresentou médias superiores em assimilação de CO2 (A), eficiência de carboxilação (A/Ci) e taxa de crescimento relativo (TCR). As doses de nitrogênio influenciaram positivamente A/Ci, teores de clorofila, área foliar (de limbo e total) e concentração foliar de nitrogênio, embora não diferindo entre 10 ou 15g de N planta-1. Para o acúmulo dos nutrientes Ca, Mg, S, Fe e Zn, a adubação nitrogenada não representou maiores teores foliares, embora isto possa estar relacionado a um efeito de diluição.

En raison de leur rôle central dans la physiologie et le développement des plantes, les phythormones ont depuis longtemps été considérées comme des médiateurs chimiques déterminants dans la capacité des insectes à contrôler leur environnement végétal. Les mécanismes permettant aux insectes de manipuler la balance phytohormonale permettant ainsi la régulation des apports nutritifs et la modulation des défenses végétales demeurent néanmoins pour la plupart inconnus, en particulier pour les insectes galligènes et mineurs de feuilles. L’objectif de ma thèse visait à caractériser les capacités de modulation phytohormonale par les insectes manipulateurs de plantes avec un accent particulier sur le lépidoptère mineur de feuille Phyllonorycter blancardella. Nous avons ainsi développés une caractérisation spatio-temporelle de la réponse des plantes aux attaques des larves mineuses au niveau moléculaire et biochimique. Une comparaison entre différents systèmes biologiques nous a permis d’évaluer les similitudes entre les stratégies adoptées par les les insectes galligènes et les insectes mineurs de feuilles, d’identifier l’origine possible des phytohormones impliquées dans la manipulation de la plante et le rôle des bactéries endosymbiotiques d’insectes dans ces interactions. / Because phytohormones lie at the very core of molecular mechanisms controlling the plant physiology and development, they have long been hypothesized to be involved in insect-induced plant manipulations. Insects are using phytohormones to manipulate their host plants for their own benefit, regulating nutrient provisioning and plant defenses. However, a mechanistic understanding of how phytohormones operate in plant reconfigurations by plant-manipulating insects, especially by gall-inducing and leaf-minging insects, is lacking. The objective of my Ph.D. was to provide an extensive characterization of how plant-manipulating insects modulate the plant global hormonal balance with a specific focus on the leaf-mining moth Phyllonorycter blancardella. We thus developed a time course characterization of plant transcriptomic and biochemical responses following attack by leaf-mining larvae. A comparative analysis between different biological systems allowed us to estimate similarities in strategies developed leaf-mining and gall-inducing insects, to identify the possible origin of phytohormones involved in the plant manipulation and to estimate the role of insect endosymbiotic bacteria in these interactions.

Diverse and ecologically important microbial communities (microbiomes) are symbiotic within marine sponges. In this study, the microbiome of Amphimedon compressa from three sample locations (Broward and Dade Counties, Southeast Florida, USA and the Southern Caribbean, Bocas del Toro, Panama) is characterized using 16S rRNA Illumina sequencing. The predominant taxa are Proteobacteria and Cyanobacteria, as expected for Low Microbial Abundance sponges, accounting for over 53% of the total microbiome community. The numbers of Operational Taxonomic Units (OTUs) decrease from Broward County (2,900) to Dade County (2,300) and then Bocas del Toro (1,200). The correlates to a decreasing north-south gradient of sponge microbiome richness and diversity. Sponge microbiome richness and Alpha diversity are nearly identical from the two closest locations (37 km), both in Southeast Florida (Tukey HSD/ANOVA; p=0.999). However Panama sponge microbiome richness and Alpha diversity are distinctly lower, with the primary driver being distance, ~1,850 km from Southeast Florida. Abiotic factors driving this trend of decreased richness and diversity include increased temperature, and deceased salinity in relation to precipitation-based seasons. Sponge microbiome Beta diversity as determined by Bray-Curtis Dissimilarity and Non-Metric Multidimensional Scaling documents the clustering of Panama samples as distinct from the Broward and Dade County samples. In a seasonal comparison, Broward County sponge microbiome richness (p=0.026, r2=0.92) and Alpha diversity (p=0.007, r2=0.98) are significantly different, documenting robust effects of temperature. This comparison confirms lowest microbiome OTU diversity in the season with highest precipitation and highest temperatures of 29.8 °C. These results are consistent with prior studies that report decreasing microbiome OTU richness and diversity under conditions of environmental stress such as decreased salinity and increased temperatures.

Symbiosis between bacteria and single celled eukaryotes, such as ciliates and amoebae, is a widespread phenomenon. In the past years, the number of species characterizations of symbiotic bacteria, has constantly grown. Especially members of the order Rickettsiales (Alphaproteobacteria), which comprises obligate intracellular human and animal pathogens as well as other non-pathogenic intracellular species, got into the focus. However, biodiversity of both, protists and associated bacterial symbionts, is still largely underestimated. Thus, I hypothesised that different kind of bacteria occur naturally in the ciliate Paramecium, that paramecia can serve as potential reservoir for human and animal pathogens, and that the species distribution of Paramecium and its endosymbionts shows geographical patterns. In the present thesis, I provide data on the distribution and diversity of Paramecium species and their endosymbionts isolated from freshwater and brackish habitats from North and South America. The most frequent species were Paramecium biaurelia, Paramecium caudatum and Paramecium multimicronucleatum for the North American samples as well as P. multimicronucleatum and Paramecium calkinsi for the South American samples indicating geographical differences in the species distribution of Paramecium between the two continents. Monoclonal cultures were screened for the presence of endosymbionts by fluorescence in situ hybridization (FISH) using universal and specific probes. I detected bacterial endosymbionts belonging to different taxa of Rickettsiales sensu lato in Paramecium-cultures isolated from eight different habitats. Moreover, I characterized the four novel endosymbiont species “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” and “Candidatus Jurandia parameciophila”, respectively, presenting detailed descriptions. Thus, I did not only detect various bacterial symbionts in different host species from two continents, but I also identified bacteria related to human and animal pathogens. Moreover, phylogenetic information provided by the four novel bacterial species helped me to revise the taxonomic organisation of the order Rickettsiales, which I split into the two orders Rickettsiales sensu stricto and Holosporales ord. nov. The importance and severity of a symbiosis for its host can be estimated, among others, by studying the symbionts transmission modes. Most host-symbiont systems in nature show a mixed mode of transmission, even if one transmission type occurs only rarely. Moreover, evolution of exclusively vertically or horizontally transmitted symbionts is scarce. However, circumstances for horizontal transmission of many symbiont species so far described as vertically transmitted, are completely unknown and still need to be revealed. Therefore, I assumed that both, vector organisms and environmental stress, can trigger horizontal transmission. I performed different sets of infection experiments using vector organisms and environmental stress factors as driving forces to trigger horizontal transmission. In experiments focused on vector-induced transmission, I was not able to confirm vector properties of Holospora towards Caedibacter, as described towards food bacteria. However, I rarely observed vector-independent horizontal transmission of Caedibacter taeniospiralis towards symbiont-free Paramecium species. The here tested genotypes of C. taeniospiralis might still take advantage of additional horizontal transmission, even if it occurs rarely. In addition, I tested the effect of salinity and temperature stress on the transmission of the recently described bacterial symbionts “Candidatus Megaira polyxenophila” and “Ca. Bealeia paramacronuclearis” as potentially interesting representatives. The involved symbiont and host genotypes differed in their response to the induced environmental stress. Results of horizontal transmission turned out to be ambiguous. While temperature stress did not show any effect on transmission, salinity stress could possibly induce horizontal transmission of “Ca. Megaira polyxenophila” at slightly increased salinity concentrations in regard to normal conditions indicating the significance of environmental stress for the selection of the preferred transmission mode. / Symbiosen zwischen Bakterien und einzelligen Eukaryoten wie Ciliaten und Amöben sind weitverbreit. In den vergangenen Jahren stieg die Anzahl der Artbeschreibungen symbiotisch lebender Bakterien stetig an. Besonders Vertreter der Ordnung Rickettsiales (Alphaproteobacteria), welche obligat intrazelluläre Menschen- und Tierpathogene sowie andere nicht-pathogene, intrazelluläre Arten beinhaltet, rückten in den Fokus. Die Biodiversität von Protisten und mit ihnen assoziierte, bakterielle Symbionten wird jedoch weitestgehend unterschätzt. Daher untersuchte ich, welche verschiedenen Bakterienarten im Ciliaten Paramecium vorkommen, ob Paramecien als potentielles Reservoir für menschen- und tierpathogene Bakterien dienen können und ob die Artverbreitung von Paramecium und seiner Symbionten geographische Muster aufweist. In der vorliegenden Arbeit, präsentiere ich Verbreitungs- und Diversitätsdaten von Paramecium-Arten sowie deren Endosymbionten, welche aus nord- und südamerikanischen Süß- und Brackwasserproben isoliert wurden. Die am häufigsten erfassten Arten in nordamerikanischen Proben waren Paramecium biaurelia, Paramecium caudatum und Paramecium multimicronucleatum, sowie P. multimicronucleatum und Paramecium calkinsi in südamerikanischen Proben. Die identifizierten Arten weisen auf geographische Unterschiede in der Artverteilung der beiden Kontinente hin. Monoklonale Massenkulturen wurden unter Verwendung universeller und spezifischer Sonden mittels Fluoreszenz in situ Hybridisierung auf das Vorhandensein von Endosymbionten überprüft. Dabei entdeckte ich bakterielle Endosymbionten verschiedener Rickettsiales sensu lato-Vertreter in Paramecienkulturen aus acht verschiedenen Habitaten. Darüber hinaus charakterisierte ich die vier neuen Endosymbiontenarten “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” und “Candidatus Jurandia parameciophila” und lieferte detaillierte Artbeschreibungen. Somit erfasste ich nicht nur verschiedene Symbionten in unterschiedlichen Paramecium-Arten der beiden Kontinente, sondern identifizierte auch Bakterien, welche nahe mit relevanten Pathogenen verwandt sind. Die phylogenetischen Informationen, die mir diese vier neuen Bakterienarten lieferten, halfen mir die taxonomische Organisation der Ordnung Rickettsiales zu revidieren, welche ich in die zwei Ordnungen Rickettsiales sensu stricto und Holosporales ord. nov. aufspaltete. Die Bedeutung einer Symbiose für seinen Wirt kann unter anderem durch die Erforschung der Übertragungswege abgeschätzt werden. Die meisten natürlichen Wirt-Symbionten-Systeme zeigen gemischte Übertragungswege, auch wenn eine der Übertragungsarten nur wenig genutzt wird. Ferner tritt eine Evolution hin zu sich ausschließlich vertikal oder horizontal übertragenden Symbionten selten auf. Die Bedingungen, unter denen eine horizontale Übertragung der meisten sich als vertikal übertragend beschriebenen Symbionten stattfindet, sind unbekannt und müssen nach wie vor untersucht werden. Deshalb legte ich meiner Arbeit die Annahme zugrunde, dass sowohl Vektororganisen als auch Umweltstress eine horizontale Übertragung begünstigen können. Ich führte verschiedene Infektionsexperimente durch, in denen ich die Effekte von Vektororganismen und verschiedener abiotischer Stressfaktoren als potenzielle Auslöser für eine horizontale Übertragung untersuchte. In den Experimenten, die sich mit einer Vektor-induzierten Übertragung beschäftigten, konnte ich keine möglichen Vektoreigenschaften von Holospora gegenüber Caedibacter nachweisen, wie sie gegenüber Futterbakterien bekannt sind. In seltenen Fällen beobachtete ich jedoch eine Vektor-unabhängige, horizontale Übertragung von Caedibacter taeniospiralis auf Symbionten-freie Paramecium-Arten. Die hier getesteten C. taeniospiralis-Genotypen sind möglicherweise immer noch dazu in der Lage, die Vorteile einer zusätzlichen horizontalen Übertragung zu nutzen. Zusätzlich testete ich die Effekte von Salz- und Temperaturstress auf die Übertragung der kürzlich beschriebenen, bakteriellen Symbionten “Candidatus Megaira polyxenophila” und “Ca. Bealeia paramacronuclearis” als potentiell interessante Vertreter. Die getesteten Symbionten- und Wirtsgenotypen unterschieden sich hinsichtlich ihrer Reaktion auf den ausgelösten abiotischen Stress. Die erzielten Ergebnisse zur horizontalen Übertragung erscheinen jedoch nicht eindeutig. Während Temperaturstress keine Effekte auf die Übertragung hatte, führte leichter Salzstress zu einer möglichen horizontalen Übertragung von “Ca. Megaira polyxenophila”, was auf die Bedeutung abiotischer Stressfaktoren bei der Wahl des bevorzugten Übertragungsweges hinweist. / Le simbiosi fra batteri ed eucarioti unicellulari, come i ciliati e le amebe, è un fenomeno molto diffuso. Negli anni passati, il numero di descrizioni di specie di batteri simbionti è cresciuto costantemente. In particolare, sono venuti alla ribalta i membri dell’ordine Rickettsiales (Alphaproteobacteria), che comprende patogeni intracellulari obbligati umani e di animali, oltre ad altre specie di batteri intracellulari non patogeni. Comunque, la biodiversità dei protisti e dei batteri simbionti a loro associati è ancora largamente sottostimata. Perciò, è stato ipotizzato che ci siano diversi tipi di batteri che vivono in associatione con il ciliato Paramecium in natura, che I parameci possano fungere da possibili serbatoi naturali per patogeni umani e animali, e che la distribuzione delle specie di Paramecium e dei suoi simbionti presenti dei pattern geografici. Nel presente lavoro di tesi, sono presentati dati sulla distribuzione e diversità di specie del genere Paramecium e dei loro batteri endosimbionti, isolate da ambienti dulciacquicoli e salmastri dal nordamerica e sudamerica. Le specie più frequenti sono state Paramecium biaurelia, Paramecium caudatum e Paramecium multimicronucleatum per i campioni nordamericani, e P. multimicronucleatum e Paramecium calkinsi per i campioni sudamericani, il che indica differenze geografiche nella distribuzione della specie di Paramecium fra I gue continenti. Colture monoclonali sono state saminate per la presenza di endosimbionti tramite ibridazione in situ a fluorescenza (FISH), impiegando sonde universali e specifiche. Sono stati rilevati batteri endosimbionti appartenenti a diversi taxa di Rickettsiales sensu lato in colture di paramecio isolate da otto diversi habitat. Inoltre, sono state caratterizzate quattro nuove specie di endosimbionti, rispettivamente, “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” e “Candidatus Jurandia parameciophila”), fornendo descrizioni dettagliate. Perciò non solo sono stati caratterizzati diversi batteri simbionti in differenti specie di ospiti da due continenti, ma sono anche stati identificati batteri affini a patogeni umani e animali. I dati filogenetici forniti dalle quattro nuove specie mi hanno permesso di revisionare l’organizzazione tassonomica dell’ordine Rickettsiales, che è stato diviso nei due ordini Rickettsiales sensu stricto e Holosporales ord. nov. L’importanza e la severità di una simbiosi per l’ospite può essere stimata studiando le modalità di trasmissione del simbionte. La maggior parte dei sistemi ospite-simbionte in natura presentano una modalità mista di trasmissione, anche se una tipo di trasmissione avviene solo occasionalmente. Inoltre, l’evoluzione di simbionti trasmessi eclusivamente per via verticale o orizzontale è scarsa. Comunque, le circostanze per la trasmissione orizzontale di molti simbionti finora descritti come trasmessi orizzontalmente sono completamente sconosciute, e necessitano di essere rivelate. Perciò, è stato ipotizzato che sia organismi vettori che stress ambientali possano indurre la trasmissione orizzontali. Sono stati eseguiti diversi set di esperimenti di infezione, impiegando organismi vettori e fattori di stress ambientali come agenti che potessero indurre tramissione orizzontale. In esperimenti incentrati sulla trasmissione indotta da vettori, non è stato possibile confermare capacità di vettore da parte di Holospora nei confronti di Caedibacter, come invece descritto nei confronti di batteri a vita libera. Comunque, in rari casi è stata osservata trasmissione orizzontale di Caedibacter taeniospiralis verso specie di Paramecium indipendente dal vettore. I genotipi testati di C. taeniospiralis potrebbero comunque trarre un vantaggio dalla possibilità alternativa di trasmissione orizzontale, anche se questa avviene solo di rado. Inoltre, è stato testato l’effetto dello stress da salinità e temperatura sulla trasmissione dei batteri simbionti recentemente descritti “Candidatus Megaira polyxenophila” e “Ca. Bealeia paramacronuclearis” come rappresentanti potenzialmente interessanti. I genotipi coinvolti di ospiti e simbionti hanno risposto diversamente a fattori indotti di stress ambientale. I risultati sono stati ambigui in termini di trasmissione orizzontale. Mentre lo stress da temperatura non ha mostrato un effetto sulla trasmissione, è stata rilevata una possibile induzione della trasmissione di “Ca. Megaira polyxenophila” in presenza di basso stress salino rispetto alle condizioni normali, indicando l’importanza degli stress ambientali per la selezione della modalità di trasmissione.