Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-06-29T19:03:41Z No. of bitstreams: 1 2015 - Francine Yuriko Otsuka Rocha.pdf: 2503949 bytes, checksum: b9cea1d05fae4647a2e2459b6401ecc0 (MD5) / Made available in DSpace on 2017-06-29T19:03:41Z (GMT). No. of bitstreams: 1 2015 - Francine Yuriko Otsuka Rocha.pdf: 2503949 bytes, checksum: b9cea1d05fae4647a2e2459b6401ecc0 (MD5) Previous issue date: 2015-09-09 / CAPES / The fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici is a disease of economic importance to tomato crop (Lycopersicon esculentum Mill.) because of the losses caused in the production. This study aimed to select bacteria belonging to the genus Bacillus which have antagonistic activity to fungi present in tomato producing areas. It was obtained 193 bacterial isolates from tomato roots and stems. We performed the complete sequencing 16S rRNA subunit of 32 bacteria from the work collection using the 27F and Amp2 primers. These bacteria were randomly selected, and 20 bacteria were obtained from healthy tomato plants. The results showed a high similarity to species of the genus Bacillus: B. thuringiensis, B. megaterium, B. cereus, B. toyonensis, B. stratosphericus and Paenibacillus barcinonensis. Evaluation of the functional characteristics of the bacterial strains showed that 72% possess proteolytic activity, 78% have cellulolytic activity, 48% are able to solubilize inorganic phosphate, 42% solubilize zinc oxide and 40.8% are able to produce indols. Antagonism tests carried out in vitro showed that some bacterial strains reduced the mycelial growth of the three races of the fungus when subjected to direct method (parallel streaks). An experiment was conducted using the genotypes Debora Plus, Santa Clara Miss Brazil, Perinha and Carolina in order to select a sensitive tomato genotype to the three fungal races. The Perinha variety was the most susceptible for races 1, 2 and 3 and presented the highest percentage of fungal colonization. Thus, this variety was used in a greenhouse experiments to evaluate the antagonistic role of the Bacillus strains against three fungal races. The antagonistic activity of bacteria was assessed 15 days after transplanting of the tomato seedlings to contaminated substrate. The results showed that the B. stratosphericus strain contributed to control at least 93.25% of the damage caused by race 1, strains from species B. megaterium and B. thuringiensis controlled 100% the damage caused by race 2 and at least 67.35% of the disease caused by the race 3 of the fungus. The results suggest that some strains have the potential to control race 1, 2 and 3 of the F. oxysporum f. sp. lycopersici and have functional activities that may contribute to improving the growth of tomato plants. However, field studies are needed to confirm the role of these Bacillus species during the interaction with tomato plants grown in soil contaminated with the race 1, 2 and 3 of the fusarium wilt. / A murcha-de-fus?rio causada por Fusarium oxysporum f. sp. lycopersici ? uma doen?a de grande import?ncia econ?mica para a cultura do tomateiro (Lycopersicon esculentum Mill.) devido as perdas que ocasiona na produ??o. O presente estudo teve como objetivo selecionar bact?rias pertencentes ao g?nero Bacillus que apresentam atividade antag?nica para aplica??o em ?reas produtoras de tomate infestadas pelo fungo. Foram obtidos 193 isolados de bact?rias de ra?zes e hastes de tomateiro. Foi realizado o sequenciamento completo da subunidade 16S RNAr de 32 isolados da cole??o de trabalho, utilizando os iniciadores 27F e Amp2. Essas bact?rias foram selecionadas aleatoriamente, sendo que 20 bact?rias foram obtidas de tomateiro sadio. Os resultados mostraram uma alta similaridade das estirpes com as esp?cies do g?nero Bacillus: B. thuringiensis, B. megaterium, B. cereus, B. toyonensis, B. stratosphericus, al?m da esp?cie Paenibacillus barcinonensis. Testes para avaliar as caracter?sticas funcionais das estirpes bacterianas in vitro mostraram que 72% das estirpes possuem atividade proteol?tica, 78% possuem atividade celulol?tica, 48% solubilizam fosfato inorg?nico, 42% solubilizam zinco e 40,8% s?o capazes de produzir indol. Testes de antagonismo realizados in vitro mostraram que algumas estirpes bacterianas retardaram ou reduziram o crescimento micelial das tr?s ra?as do fungo quando submetidos ao m?todo de a??o direta (estrias paralelas). Com o objetivo de selecionar um gen?tipo de tomateiro sens?vel as tr?s ra?as do fungo, foi instalado um experimento utilizando os gen?tipos D?bora Plus, Santa Clara Miss Brasil, Perinha e Carolina. A variedade Perinha foi a mais suscet?vel as ra?as 1, 2 e 3 j? que apresentou um maior percentual de coloniza??o do fungo. A atividade antag?nica das bact?rias foi avaliada 15 dias ap?s o transplantio das mudas de tomate da variedade Perinha para o substrato contaminado. Os resultados mostraram que a estirpe da esp?cie B. stratosphericus controlou cerca de 93,25% das plantas crescidas na presen?a da ra?a 1 do fungo. J? as esp?cies B. megaterium e B. thuringiensis controlaram 100% da doen?a das plantas de tomateiro cultivadas em substrato com a ra?a 2 e de 67,35% das plantas cultivadas em substrato com a ra?a 3 do fungo. Os resultados obtidos sugerem que algumas estirpes possuem potencial para controle de F. oxysporum f. sp. lycopersici ra?a 1, 2 e 3 e possuem atividades funcionais que podem contribuir para a melhoria do crescimento das plantas de tomateiro cultivadas em ?reas infestadas com o fungo. Entretanto, estudos em campo s?o necess?rios para confirmar o papel dessas esp?cies de Bacillus na intera??o com plantas de tomateiro cultivadas na presen?a das ra?as 1, 2 e 3 da murcha de fus?rio.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-21T19:29:33Z No. of bitstreams: 1 2016 - Caroline Barra Sales Khayat da Costa.pdf: 5220401 bytes, checksum: 553248261c960b6b5fb62cadb1b26ebf (MD5) / Made available in DSpace on 2018-09-21T19:29:33Z (GMT). No. of bitstreams: 1 2016 - Caroline Barra Sales Khayat da Costa.pdf: 5220401 bytes, checksum: 553248261c960b6b5fb62cadb1b26ebf (MD5) Previous issue date: 2016-07-26 / CAPES / One of the most known and important process in nature is the biological nitrogen fixation (FBN). Few classes of microorganisms perform this process; named diazotrophics that associated with plants is an important alternative to enhance nitrogen nutrition in agricultural systems. Diazotrophic bacterial population in association with sugarcane improves the yield due to its ability to colonize the inner roots of the host plant and do not provoke any pathogenicity signal. Quantitative analyzes have indicated that selection of combinations of endophytic diazotrophic strains when associated with sugarcane varieties can improve the yield and therefore varieties of sugarcane may be better exploited in order to benefit this association for agricultural purposes. Thus, this work proposes to monitor the establishment of diazotrophic bacteria present in the inoculant of Embrapa, during the initial development of sugarcane plants. Micro-propagated sets of the sugarcane variety RB867515 were inoculated individually and with a mixture of five nitrogen fixing bacteria strains that compose the sugarcane inoculant: Nitrospirillum amazonense (CBAmC) Paraburkholderia tropica (Ppe8) Gluconacetobacter diazotrophicus (Pal5) Herbaspirillum seropedicae (HRC54) and Herbaspirillum rubrisubalbicans (HCC103). Plants were grown in a greenhouse and harvested 30 days after planting. Fresh root material was separated for evaluation of the establishment of the strains by bacterial counting using the technique of Most Probable Number (MPN), absolute quantification by Real Time PCR (qPCR), besides the identification of the inoculated strains through analysis of the rDNA profile of the pellicle formed in the respective semisolid media using the BOX-PCR technique. The results of bacterial count diazotrophic present in the roots by NMP showed that the treatments inoculated in the control (native population) showed higher population of bacterial cells around 105 cells by fresh root mass, but not statistically significant. The methodology qPCR permitted quantitation of the number of 16S rDNA copies in the order of 105 bacterial cells g - 1 fresh weight root, showing that there were differences in the population of endophytic species that colonize the roots of sugarcane. The profiles of BOX -PCR of the respective pellicles formed in semisolid media did not show high similarity (> 80 %) with the profile of the species inoculated for most of the treatments. The results obtained indicate that the qPCR technique showed the establishment of some of the inoculated strains in sugar cane buds while the NMP technique showed no significant difference between treatments inoculated and non-inoculated possibly due to the lower sensitivity of the method. / Um dos processos mais conhecidos e importantes na natureza ? a fixa??o biol?gica de nitrog?nio atmosf?rico (FBN). Este processo ? realizado por algumas classes de microrganismos, denominados diazotr?ficos, que associados com esp?cies vegetais s?o uma alternativa importante para aprimorar a nutri??o nitrogenada nos sistemas agr?colas. As popula??es de bact?rias fixadoras de nitrog?nio atmosf?rico quando associadas ? cultura da cana-de-a??car melhoram a produ??o e, ao colonizarem o interior das ra?zes promovem benef?cios ?s plantas hospedeiras. An?lises quantitativas t?m indicado que a sele??o das combina??es de estirpes de bact?rias diazotr?ficas endof?ticas e variedades de cana-de-a??car podem ser melhor exploradas com o objetivo de aperfei?oar a associa??o para finalidades agr?colas. Assim, o presente trabalho prop?e monitorar o estabelecimento das bact?rias diazotr?ficas presentes no inoculante para cana-de- a??car da Embrapa, durante o desenvolvimento inicial planta. Toletes propagados da variedade RB867515 de cana-de-a??car foram inoculados individualmente e com a mistura das cinco estirpes de bact?rias diazotr?ficas: Nitrospirillum amazonense (CBAmC), Paraburkholderia tropica (PPe8), Gluconacetobacter diazotrophicus (Pal5), Herbaspirillum seropedicae (HRC54) e Herbaspirillum rubrisubalbicans (HCC103). As plantas foram crescidas em casa de vegeta??o e coletadas 30 dias ap?s o plantio, sendo separado o material vegetal para avalia??o do estabelecimento das estirpes pela t?cnica do N?mero Mais Prov?vel (NMP) e quantifica??o absoluta por PCR em Tempo Real (qPCR). Adicionalmente foi feita a identifica??o das estirpes inoculadas atrav?s da an?lise do perfil de rDNA das pel?culas bacterianas formadas nos respectivos meios semiss?lidos pela t?cnica de BOX-PCR. Os resultados obtidos da contagem de bact?rias diazotr?ficas presente nas ra?zes por NMP mostrou que os tratamentos inoculados em rela??o ao controle (popula??o nativa), apresentaram uma maior popula??o de c?lulas bacterianas em torno de 105 c?lulas por massa fresca de raiz, por?m n?o significativa. A metodologia de qPCR permitiu a quantifica??o do n?mero de c?pias do 16S rDNA, da ordem de 105 c?lulas bacterianas g-1 massa fresca de raiz, mostrando que houve diferen?a na popula??o das esp?cies diazotr?ficas endof?ticas que colonizam as ra?zes de cana-de-a??car. Os perfis obtidos por BOX-PCR das pel?culas formadas nos respectivos meios semiss?lidos n?o mostraram alta similaridade (>80%) com o perfil das esp?cies inoculadas para a maioria dos tratamentos inoculados. Os resultados obtidos indicam que a t?cnica de qPCR permitiu mostrar o estabelecimento de algumas estirpe do inoculante nos toletes de cana-de a??car enquanto que a t?cnica de NMP n?o mostrou diferen?a significativas entre os tratamentos inoculados e n?o inoculados possivelmente pela menor sensibilidade da metodologia.

Made available in DSpace on 2014-12-17T15:30:59Z (GMT). No. of bitstreams: 1 EmanuelleDVD_DISSERT.pdf: 1398010 bytes, checksum: 85e1d1f82b33da3e697eacbf6655c267 (MD5) Previous issue date: 2011-08-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Photodynamic therapy (PDT) has been proposed as an alternative method for the treatment of biofilm-dependent oral diseases like dental caries. This therapy consists of simultaneous action of a visible light (L) and a photosensitizer (FS) in the presence of oxygen, which leads to production of different reactive oxygen species that can interact with the bacterial cell components, and promote cell death. This study aims to evaluate the antimicrobial action of PDT on oral bacteria in suspension, as well as the formation of mono and multi-species biofilms, in vitro, from a standard strain of Streptococcus mutans (ATCC 25175) and saliva samples, respectively. The dye methylene blue (MB) and toluidine blue (TB) were used at a concentration of 100 mg/ L and activated by halogen light (600 to 750 nm) from a modified hand held photopolymerizer (Ultralux ?, Dabi Atlante, Ribeir?o Preto , S?o Paulo, Brazil.). Planktonic cultures were prepared and submitted to different experimental conditions: 1. PDT using TB 2. PDT using MB, 3. L+ FS- , 4. TB + L - ; 5. MB+ L-; 6. L- FS- (negative control) and 7. administration of 0.12% chlorhexidine digluconate (positive control) (Periogard ?, Colgate-Palmolive Company, New York, USA). The immediate and mediated action of PDT on bacterial suspensions, as well as its effect on biofilm formation were observed from the number of colony-forming units per milliliter (CFU/mL) and measures optical density (OD). The data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney test for the significance level of 5%. According to the results, the PDT showed no antibacterial action on suspensions of S. mutans, regardless of the dye used. PDT with MB activated by halogen light was able to reduce 86.6% CFU/mL multi-species planktonic cultures, however, this reduction was not significant (p > 0.05). PDT showed antibacterial effect, mediate on multi-species planktonic cultures with TB (p < 0.001) and MB (p < 0.001), activated by halogen light. PDT was able to prevent the formation of multispecies biofilm, through the activation of TB by halogen light (p = 0.01). We conclude that activation of the dye toluidine blue and methylene blue, by halogen light (PDT) showed antimicrobial activity, compared to multi-species planktonic cultures prepared from saliva samples / A terapia fotodin?mica (TFD) tem sido proposta como um m?todo alternativo para o tratamento de doen?as orais biofilme-dependentes, como a c?rie dent?ria. Tal terapia consiste na a??o simult?nea de uma fonte luminosa (FL) e de um agente fotossensibilizante (FS), na presen?a de oxig?nio tecidual, levando ? produ??o de radicais livres que ao interagirem com componentes celulares bacterianos, promovem a morte celular. Este estudo tem como objetivo avaliar a a??o antimicrobiana da TFD sobre bact?rias orais em suspens?o, bem como na forma??o de biofilmes mono e multiesp?cies, in vitro, a partir de uma cepa padr?o de Streptococcus mutans (ATCC 25175) e de amostras de saliva, respectivamente. Os corantes azul de metileno (CM) e azul de toluidina (CT) foram administrados na concentra??o de 100 mg/L e ativados por luz hal?gena (600 a 750 nm) proveniente de um aparelho fotopolimerizador modificado (Ultralux?, Dabi Atlante, Ribeir?o Preto, S?o Paulo, Brasil.). Suspens?es monoesp?cies e mistas foram preparadas e submetidas a diferentes condi??es experimentais: 1. TFD na presen?a de CT; 2. TFD na presen?a de CM; 3. irradia??o sem administra??o de qualquer corante; 4. sensibiliza??o com CT e aus?ncia de luz; 5. sensibiliza??o com CM e aus?ncia de luz; 6. aus?ncia de irradia??o e sem qualquer corante (controle negativo) e 7. administra??o de solu??o de digluconato de clorexidina a 0,12% (controle positivo) (Periogard?, Colgate-Palmolive Company, Nova York, EUA). A a??o imediata e mediata da TFD sobre as suspens?es bacterianas, bem como seu efeito sobre a forma??o de biofilme foram verificados, a partir do n?mero de unidade formadoras de col?nias por mL (UFC/mL) e de medidas de densidade ?tica (DO). Os dados obtidos foram analisados estatisticamente atrav?s dos Testes Kruskal-Wallis e Mann-Whitney, para o n?vel de signific?ncia de 5%. De acordo com os resultados, a TFD n?o apresentou a??o antibacteriana sobre as suspens?es de S. mutans, independente do corante utilizado. A TFD com CM ativado por luz hal?gena foi capaz de reduzir em 86,6% o n?mero de UFC/mL diante de suspens?es multiesp?cies, entretanto, tal redu??o n?o foi significativa (p > 0,05). A TFD apresentou efeito antibacteriano, mediato, sobre suspens?es mistas, quer na presen?a de CT (p < 0,001) ou CM (p < 0,001), ativados por luz hal?gena. A TFD preveniu a forma??o de biofilme multiesp?cie, atrav?s da ativa??o de CT por luz hal?gena (p = 0,01). Conclui-se que a ativa??o dos corantes azul de toluidina e azul de metileno, pela luz hal?gena (TFD) apresentou a??o antimicrobiana, frente a suspens?es multiesp?cies preparadas a partir de amostras de saliva, ressaltando a possibilidade da utiliza??o da terapia fotodin?mica como um m?todo coadjuvante no tratamento de les?es de c?rie dent?ria