Recent works has highlighted the contribution of microbes to animal function. In this regard, the microbial community associated with corals has become a growing field of research in order to understand how microbes contribute to the host organisms’ response to environmental changes. It has been shown that microbes associated with corals have important functions in the coral holobiont such as immunity and nutrient assimilation. However, corals are notoriously difficult to work with. To this end, the sea anemone Aiptasia is becoming a model organism for coral symbiosis. Given the importance of host-­microbiome interactions, the topic of this thesis is to assess microbial structure of Aiptasia, culture prominent bacterial members, and compare bacterial community structure to corals. Different molecular methods have been applied using 16S rRNA bacterial gene fragments to characterize the microbial composition of Aiptasia. 16S rRNA gene sequence derived from cultured bacteria was compared to 16S rRNA gene sequences retrieved from native Red Sea Aiptasia. Inter-­individual as well as methodological differences were found to account for variance in microbiome composition. However, all approaches showed a highly abundant microbial taxon belonging to the genus Alteromonas in all samples. The Alteromonas species was successfully isolated for further research targeting microbiome selection mechanisms in Aiptasia. Future investigations by using different molecular tools will help to define the functions and relationship between the Aiptasia and its complex microbiome.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-08-11T13:01:57Z No. of bitstreams: 1 PDF - Jonnathan Whiny Moraes dos Santos.pdf: 20146290 bytes, checksum: 1be326dec739963cb441a03e24a0f84c (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-08-29T15:45:18Z (GMT) No. of bitstreams: 1 PDF - Jonnathan Whiny Moraes dos Santos.pdf: 20146290 bytes, checksum: 1be326dec739963cb441a03e24a0f84c (MD5) / Made available in DSpace on 2017-08-29T15:45:18Z (GMT). No. of bitstreams: 1 PDF - Jonnathan Whiny Moraes dos Santos.pdf: 20146290 bytes, checksum: 1be326dec739963cb441a03e24a0f84c (MD5) Previous issue date: 2017-04-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Belonging to the Fabaceae family, the peanut (Arachis hypogaea) is cultivated worldwide, having great importance in the income of the cultivator. Because it is a culture that associates with bacteria that have the capacity to fix nitrogen, it has been studied in order to potentiate this symbiotic relationship and favor the increase of its production. Thus the objective of this work was to evaluate the diversity and symbiotic efficiency of the native bacteria associated with Arachis species. A collection of bacteria isolated from nodules of six species of the genus Arachis cultivated in samples of six soils from the semi - arid region was structured. The isolates were purified and evaluated for their phenotypic characteristics, and amplification of some symbiotic genes. The isolates considered as nifH, nodA and / or nodC positive we re selected for the symbiotic efficiency test, as well as for the partial sequencing reactions of the 16S rRNA gene. Bacteria selected in previous evaluations were tested in a greenhouse experiment to evaluate symbiotic efficiency. Bacteria classified as Bradyrhizobium, the 16S rRNA gene were also sequenced in reverse. In addition, the recA, nifD and nodC genes were also amplified and sequenced. For these same isolates, a BOX-PCR was performed to verify differences between these isolates. Phylogenetic trees were elaborated based on the genes gathered from the results. A collection of 365 isolates was obtained, with the majority of the isolates showing rapid growth, acidifying the culture medium, colony diameter of 1 to 2 mm, and yellowing. Of the total isolates, 253 had amplification in at least one of the evaluated symbiotic genes, 94 isolates amplified for all genes and primers evaluated. A total of 63 isolates were selected for sequencing of the 16S rRNA gene, as well as for the evaluation of symbiotic efficiency in greenhouse. From the obtained results, 45 sequences presented good quality and adequate size and were used for the comparison with those available in the EzBioCloud database. A total of 22 isolates were identified as belonging to the genus Bradyrhizobium, with 21 being classified in the clade I B. japonicum and only 1 in the clade II B. elkanii, and 23 others belonging to non-rhizobian genera. From the sequencing of the symbiotic genes, it was found that some isolates that had amplifications in the three evaluated genes, such as the isolates S2AB2, S2AD2, S2AH 9.1 and S2AH 4.1, remained in distinct groups in all three symbiotic gene trees, as well as in the 16S rRNA gene tree, confirming the low similarity of these isolates to the type strains, indicating that they are possibly isolated from new species. With the BOX-PCR it was confirmed that the isolates presented a high degree of polymorphism, and that these were actually different isolates. The isolates tested in greenhouse were able to nodulate commercial cultivar BR1. The co- inoculation with non-rhizobial bacteria may have favored some isolates in their symbiotic efficiency, mainly in the accumulation of N in plants. These results will support studies for future evaluations of the selected isolates in the recommendation of inoculants of native species for the peanut crop. Cultivated peanuts and wild species evaluated may establish symbiotic associations with native strains of rhizobia with high diversity and symbiotic efficiency. / Pertencente à família Fabaceae o amendoim (Arachis hypogaea) é cultivado mundialmente, tendo grande importância na geração de renda do produtor. Por ser uma cultura que se associa a bactérias que têm a capacidade de fixar nitrogênio, vem sendo estudada visando potencializar essa relação simbiótica e favorecer o aumento de sua produção. Dessa forma o objetivo desse trabalho foi avaliar a diversidade e a eficiência simbiótica de bactérias nativas associadas a espécies de Arachis. Foi estruturada uma coleção de bactérias isoladas de nódulos de 6 espécies do gênero Arachis cultivadas em amostras de 6 solos da região semiárida. Os isolados foram purificados e avaliados quanto às suas características fenotípicas, e amplificação de alguns genes simbióticos. Os isolados considerados nifH, nodA e/ou nodC positivos foram selecionados para o teste de eficiência simbiótica, assim como para as reações de sequenciamento parcial do gene 16S rRNA. Bactérias selecionadas nas avaliações anteriores foram testadas em um experimento em casa de vegetação para avaliar a eficiência simbiótica. As bactérias classificadas como Bradyrhizobium, o gene 16S rRNA foi sequenciado também no sentido reverso. Além disso, foram amplificados e sequenciados também, os genes recA, nifD e nodC. Para estes mesmos isolados, foi realizado um BOX-PCR, para verificar diferenças entre esses isolados. A partir desses resultados foram elaboradas as árvores filogenéticas com base nesses genes. Uma coleção de 365 isolados foi obtida sendo que a maioria dos isolados apresentaram crescimento rápido, acidificaram o meio de cultura, tinham diâmetro de colônia de 1 a 2mm, além de apresentarem coloração amarela. Do total de isolados, 253 tiveram amplificação em pelo menos uns dos genes simbióticos avaliados, 94 isolados amplificaram para todos os genes e iniciadores avaliados. Um total de 63 isolados foram selecionados para sequenciamento do gene 16S rRNA, assim como para a avaliação da eficiência simbiótica em casa de vegetação. A partir dos resultados obtidos, 45 sequências apresentaram boa qualidade e tamanho adequado e foram utilizadas para a comparação com aquelas disponíveis no banco de dados EzBioCloud. Um total de 22 isolados foram identificados como pertencentes ao gênero Bradyrhizobium, sendo 21 enquadrados no clado I B. japonicum e apenas 1 no clado II B. elkanii, e outros 23 como pertencentes a gêneros não rizobianos. A partir do sequenciamento dos genes simbióticos, verificou-se que alguns isolados que tiveram amplificações nos três genes avaliados, como os isolados S2AB2, S2AD 2, S2AH 9.1 e S2AH 4.1, permaneceram em grupos distintos em todas as três árvores dos genes simbióticos, assim como também na árvore do gene 16S rRNA, confirmando a baixa similaridade desses isolados com as estirpes tipo, indicando que são possivelmente isolados de novas espécies. Com o BOX-PCR foi confirmado que os isolados apresentavam alto grau de polimorfismo, e que estes realmente eram isolados diferentes. Os isolados testados em casa de vegetação foram capazes de nodular a cultivar comercial BR 1. A coinoculação com bactérias não rizobianas pode ter favorecido alguns isolados, em sua eficiência simbiótica, principalmente no acúmulo de N nas plantas. Estes resultados subsidiarão estudos para futuras avaliações dos isolados selecionados na recomendação de inoculantes de espécies nativas para a cultura do amendoim. O amendoim cultivado e as espécies silvestres avaliadas podem estabelecer associações simbióticas com estirpes nativas de rizóbios com elevada diversidade e eficiência simbiótica.