Search

The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178 bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2022-09-27T12:38:21Z No. of bitstreams: 1 Sueide Karina da Silva.pdf: 44277941 bytes, checksum: 5c6a9558edcba5ee36369d6f5c8326f5 (MD5) Made available in DSpace on 2022-09-27T12:38:21Z (GMT). No. of bitstreams: 1 Sueide Karina da Silva.pdf: 44277941 bytes, checksum: 5c6a9558edcba5ee36369d6f5c8326f5 (MD5) Previous issue date: 2022-08-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Rhizobia are bacteria known to infect legume roots, forming nodules, where these organisms can fix atmospheric nitrogenmore efficiently than the diazotrophic bacteria that colonize grasses. Several works report the presence of rhizobia inside the roots of grasses, as well as occur in sugarcane. It is not known whether these organisms have beneficial functions for plants, but if they can act as growth-promoting bacteria, the association with grassy rhizobia may represent a great possibility for the development of technologies based on bioinputs, or on more independent management of chemical fertilizers for the culture. The results obtained so far suggest that rhizobia and other non-nodulating bacteria phylogenetically related to them represent a group of microorganisms that should be better studied, regarding their application to cultures of the Poaceae family. The objective of this work was to prospect and characterize populations of rhizobia associated with the most used variety of sugarcane in Northeast Brazil, cultivated in soil with a long history of sugarcane exploitation, and to evaluate the effect of intercropping with nodulating legumes on these populations, through isolation, followed by genetic and functional characterization. An experiment was set up in the municipality of Carpina-PE, Zona da Mata de Pernambuco. The treatments were distributed in randomized blocks with four replications, representing by single crop and the intercropping of sugarcane with different species of grain-producing legumes (cowpea, peanut, and soybean). At 5 months after sugarcane planting, non-lignified roots were collected, which were macerated in saline solution and the suspension product was used as an inoculant in bait plant trials, using the same legumes of the consortia. The isolates obtained from the formed nodules were genetically characterized, through 16S rRNA sequencing, and phenotypically in YMA medium. The isolates were tested for their ability to promote growth and biomass production and to change morphoanatomical parameters of sugarcane roots, in a pre-sprouted seedling inoculation experiment of legume growth in a greenhouse experiment. In the first trial with bait plants, nodules were only formed in cowpea inoculated with the suspension prepared from the roots of sugarcane cultivated in consortium with that legume. Results indicated the lack of rhizobia populations capable of nodulating soybeans and peanuts in sugarcane tissues, as well as demonstrating that cowpea cultivation was necessary to stimulate the presence of their microsymbionts in grass roots, probably by stimulating populations naturally established into the soil. These nodules generated a collection of 20 authenticated rhizobia, with low phenotypic and genetic diversity, with 4 isolates classified as Bradyrhizobium and 7 as Rhizobium. Among all the isolates in the collection, 55% solubilized calcium phosphate and all were capable of producing AIA. The thickness of the canopy, cortex, and vascular system, as well as the density and length of root hairs in the sugarcane root were positively influenced by inoculation. Rizóbios são bactérias conhecidas por infectar raízes de leguminosas, formando nódulos onde fixam o N atmosférico de maneira mais eficiente que as bactérias diazotróficasque colonizam as gramíneas. Diversos trabalhos relatam a presença de rizóbios no interior das raizes de gramíneas, assim como a cana-de-açúcar. Não se sabe se esses organismos exercem funções benéficas para as plantas, mas, se forem capazes de atuar como bactérias promotoras de crescimento, essa associação rizóbios-gramíneas pode representar uma possibilidade de desenvolvimento de tecnologias baseadas em bioinsumos, ou em manejos mais independentes de fertilizantes químicos. Os resultados obtidos até o momento sugerem que rizóbios e outras bactérias não nodulantes filogeneticamente relacionadas com eles representam um grupo de micro-organismos que deve ser mais bem estudado no sentido da sua aplicação para culturas da família Poaceae. O objetivo deste trabalho foi prospectar e caracterizar populações de rizóbios associados à variedade de cana-de-açúcar RB 92579, a mais utilizada no Nordeste do Brasil, cultivada em solo com longo histórico de exploração canavieira, e avaliar o efeito do consórcio dessa gramínea com leguminosas nodulantes sobre essas populações, por meio de isolamento, seguido de caracterização genética e funcional. Foi montado um experimento no município de Carpina-PE, Zona da Mata de Pernambuco, com tratamentos distribuídos em blocos casualizados com quatro repetições, representando o cultivo solteiro e o consórcio da cana-de-açúcar com diferentes espécies de leguminosas produtoras de grãos (feijão-caupi, amendoim e soja). Aos 5 meses após o plantio da cana foi realizada a coleta de raízes jovens, que foram maceradas em solução salina, onde o produto suspenso foi utilizado como inoculante em ensaios de plantas iscas, utilizando as mesmas leguminosas dos consórcios. Os isolados obtidos a partir dos nódulos formados foram caracterizados geneticamente, por meio do sequenciamento do 16S rRNA, e fenotipicamente em meio Yeast mannitol agar (YMA). Os isolados foram testados quanto à capacidade de promoção de crescimento, produção de biomassa e de alterar parâmetros morfoanatômicos de raízes da cana-de-açúcar, em experimento de inoculação de mudas pre-brotadas. Também foi testada a capacidade dos isolados em promover a promoção de crescimento de leguminosas, em experimento em casa de vegetação. No primeiro ensaio com plantas-iscas, só houve formação de nódulos em feijão-caupi inoculado com a suspensão preparada a partir das raízes de cana cultivada em consórcio com esta leguminosa. Este resultado indica a inexistência de populações de rizóbios com capacidade de nodular a soja e o amendoim nos tecidos da cana, além da necessidade do cultivo do feijão-caupi para estimular a presença de seus microssimbiontes nas raízes da gramínea, provavelmente por estimular as populações naturalmente estabelecidas no solo. Esses nódulos geraram uma coleção de 20 rizóbios autenticados, com baixa diversidade fenotípica e genética, sendo 4 isolados classificados como Bradyrhizobium e 7 como Rhizobium. Entre todos os isolados da coleção, 55% solubilizaram fosfato de cálcio e todos foram capazes de produzir ácido indolacético. A espessura do velame, do córtex e do sistema vascular, bem como a densidade e comprimento de pêlos radiculares da raiz da cana-deaçúcar foram influenciados positivamente com a inoculação. Dessa forma, a inoculação com rizóbios provenientes de raiz de cana-de-açúcar é promissora para um cultivo mais sustentável da cana-de-açúcar.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2022-10-18T17:54:16Z No. of bitstreams: 1 2021 - J?lia Ferreira Xavier.pdf: 1751247 bytes, checksum: ad39303a61045be0a5da3f507089b30a (MD5) Made available in DSpace on 2022-10-18T17:54:16Z (GMT). No. of bitstreams: 1 2021 - J?lia Ferreira Xavier.pdf: 1751247 bytes, checksum: ad39303a61045be0a5da3f507089b30a (MD5) Previous issue date: 2021-10-08 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior CNPq - Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico FAPERJ - Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do Rio de Janeiro Most plant species, especially crops, do not tolerate high salt concentrations. However, halophytic plants are adapted to saline soils. In the rhizosphere of these plants, associations with microorganisms can promote plant growth and increase resistance to salt stress. Therefore, this work aimed to isolate and identify bacteria from the rhizosphere of halophyte plants from different saline environments in the state of Rio de Janeiro and to evaluate their capacity to promote growth of rice. Samples of rhizospheric soils from halophytic plants Salicornia gaudicahudiana, Salicornia fruticosa, Blutaparon portulacoides, Sporobolus virginucus, and Cyperus ligularis were collected. Culture media amended with 1%, 5%, 15%, 20%, and 25% of NaCl were used for bacterial isolation. The bacterial strains were classified according to growth at different NaCl concentrations and were identified by Matrix Assisted Laser Desorption Ionization Time Of Flight/ Mass Spectrometry (MALDI-TOF MS) and by rrs gene sequencing. Subsequently, the potential of nine strains of halotolerant bacteria belonging to the genus Pseudomonas and Bacillus in promoting rice growth at concentrations of 50 mM and 200 mM was analyzed. Thus, germination percentage, total root length, root volume, root surface area, number of tips, and total shoot length were analyzed. A total of 315 bacteria classified as non-halophilic (99/315), halotolerant (171/315) and moderately halophilic (32/315) were isolated. Of the isolated bacteria, 286 were analyzed by the proteomic technique MALDI-TOF MS and classified at the genus level 57% (165/286). For those that could not be identified by MALDI TOF-MS, selected isolates were identified by 16S rRNA gene (rrs) gene sequencing. The inference of the genus and/or species was possible in 97.7% (42/43) of the isolates. The most abundant genera identified were Pseudomonas, Ochrobactrum, and Bacillus. At the optimal salt concentration of 50 mM NaCl, the isolates P51 and P164, belonging to the genera Pseudomonas, provided an increase in most parameters related to rice seed germination analyzed, as compared to the non-inoculated treatment. Isolates B231, B67, and B143 related to the genus Bacillus promoted root elongation and a higher number of tips. At the stressing concentration of 200 mM, isolates B231 and B294, related to Bacillus provided an increase in the total length of roots compared to the non-inoculated treatment. These results confirm that the rhizosphere of halophyte plants represents a promising environment for the isolation of halotolerant and halophilic bacteria and that halotolerant bacteria from the genera Pseudomonas and Bacillus can promote the germination and initial development of rice seeds in the presence or absence of salt stress. A maioria das esp?cies vegetais, principalmente culturas agr?colas, n?o toleram altas concentra??es de sais. No entanto, plantas do grupo hal?fitas s?o adaptadas a solos salinos. Na rizosfera das plantas hal?fitas ocorrem associa??es com microrganismos que auxiliam o crescimento vegetal aumentando a resist?ncia ao estresse salino. Desse modo, o objetivo do trabalho foi isolar e identificar bact?rias da rizosfera de plantas hal?fitas de diferentes ambientes salinos no estado do Rio de Janeiro e avaliar sua capacidade de promo??o de germina??o e crescimento de sementes de arroz. Foram coletadas amostras de solos rizof?ricos das plantas hal?fitas Salicornia gaudicahudiana, Salicornia fruticosa, Blutaparon portulacoides, Sporobolus virginucus e Cyperus ligularis encontradas em ambientes costeiros, como mangue e salinas. Para o isolamento das bact?rias foram utilizados meios de cultura acrescidos de 1%, 5%, 15%, 20% e 25% de NaCl. As bact?rias isoladas foram classificadas de acordo o crescimento em diferentes concentra??es NaCl e foram identificadas pela t?cnica Matrix Assisted Laser Desorption Ionization Time Of Flight/ Mass Spectrometry (MALDI-TOF MS) e pelo sequenciamento do gene rrs que codifica o rRNA 16S. Foi avaliado o potencial de promo??o de crescimento do arroz na concentra??o de 50 mM e 200 mM de NaCl de nove estirpes de bact?rias halotolerantes pertencentes aos g?neros Pseudomonas e Bacillus. Foram analisados a porcentagem de germina??o, o comprimento total da raiz, volume da raiz, superf?cie de contato radicular, n?mero de ?pices e comprimento total da parte a?rea. Foram isoladas um total de 315 bact?rias classificadas como n?o halof?licas (99/315), halotolerantes (171/315) e halof?licas moderadas (32/315). Destas, 286 foram analisadas pela t?cnica prote?mica MALDI-TOF MS, sendo 57% (165/286) identificadas em n?vel seguro para g?nero. Dentre as bact?rias n?o identificadas pela t?cnica MALDI TOF-MS, foram selecionados isolados halotolerantes e halof?licos moderados para a identifica??o pelo sequenciamento do gene rrs. A infer?ncia do g?nero foi poss?vel em 97,7% (42/43) dos isolados. Os g?neros mais abundantes identificados foram Pseudomonas, Ochrobactrum e Bacillus. Na concentra??o de 50 mM de NaCl, que se mostrou ideal para a germina??o das sementes de arroz, os isolados P51 e P164, relacionadas ao g?nero Pseudomonas, proporcionaram aumento da maioria dos par?metros analisados em compara??o ao tratamento n?o inoculado. Nessa concentra??o, os isolados B231, B67 e B143, pertencentes ao g?nero Bacillus, promoveram alongamento radicular e maior n?mero de ?pices. J? a 200 mM, concentra??o inibit?ria do processo germinativo, os isolados B231 e B294, pertencentes ao g?nero Bacillus, proporcionaram aumento no comprimento total das ra?zes em compara??o ao tratamento n?o inoculado. Estes resultados confirmam que a rizosfera de plantas hal?fitas representa um ambiente promissor para o isolamento de bact?rias halotolerantes e halof?licas, e que isolados de Pseudomonas e Bacillus halolerantes podem promover o crescimento de plantas de arroz na presen?a ou aus?ncia de estresse salino.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2022-04-13T19:06:35Z No. of bitstreams: 1 2019 - Camila Costa de Oliveira.pdf: 1061622 bytes, checksum: 7ce3513cc84d5e88a63c254d02e32daf (MD5) Made available in DSpace on 2022-04-13T19:06:35Z (GMT). No. of bitstreams: 1 2019 - Camila Costa de Oliveira.pdf: 1061622 bytes, checksum: 7ce3513cc84d5e88a63c254d02e32daf (MD5) Previous issue date: 2019-08-16 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior CNPq - Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico FAPERJ - Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do Rio de Janeiro Antimicrobial resistance has emerged globally as one of the greatest threats to public health, mainly due to the widespread use of antimicrobial in humans and animal production. The use of animal manure provides benefits to the soil, besides being an alternative to the discharge of this residue in the environment, since animal production is an expressive activity of the Brazilian agribusiness. However, it can increase bacteria and antimicrobial resistance genes and promote its dissemination to commensal and pathogenic bacteria of humans and animals. Strategies to manage these residues, such as composting, are important tools to ensure the safety of their use as organic fertilizer. In this context, the aim of this study was to evaluate the presence of antimicrobial resistance genes in soils of agricultural crops treated with fresh poultry manure and legal reserve areas near these production areas in Nova Friburgo, RJ, in order to clarify the role of these environments as a reservoir and source of dissemination of antimicrobial resistance. It was also evaluated the effect of the composting process of poultry manure on the prevalence of resistance genes. After the total DNA extraction of the soil samples, the detection of the resistance genes to ?-lactamics, colistin and sulphonamides (blaampC, mcr-1, sul1 and sul2, respectively) was performed using PCR (Polymerase Chain Reaction), followed by a correlation analysis of the presence of the genes in relation to the physical-chemical attributes of soils. The relative abundance of the mcr-1 gene was determined by the qPCR (Quantitative Polymerase Chain Reaction) technique. The presence of resistance genes was also evaluated at 0, 30, 60, 90 and 120 days of poultry manure composting. There was a significant difference (P < 0.05) between the proportions of genes sul1 and sul2 in the areas of agricultural production and legal reserve, being predominant in the agricultural areas. The mcr-1 gene was detected in all soil samples. The log of relative abundance of the mcr-1 gene ranged from-1.76 (1.81 x 10-? copies of mcr-1/16S rDNA) to -3.12 (7.67 x 10-4 copies of mcr-1/16S rDNA). The blaampC gene was not detected after 30 days of composting. Otherwise, the genes sul1, sul2 and mcr-1 were detected up to 120 days of composting. These results reinforce the importance of studies that aimed at elucidating the pathways for the dissemination of antimicrobial resistance genes in agricultural production areas, as well as the factors that interfere in the persistence and dissemination of these genes in the environment, in order to subsidize the implementation of management practices that reduce the risk of spreading resistance, which is a potential threat to public health. A resist?ncia antimicrobiana tem emergido globalmente como uma das maiores amea?as para sa?de p?blica, principalmente devido ao uso generalizado de antimicrobianos em humanos e na produ??o animal. O uso de esterco animal proporciona benef?cios para o solo, al?m de ser uma alternativa para a disposi??o deste res?duo no ambiente, uma vez que a produ??o animal ? uma atividade expressiva do agroneg?cio brasileiro. Por?m, pode incrementar bact?rias e genes de resist?ncia a antimicrobianos e favorecer sua dissemina??o para bact?rias patog?nicas e comensais de humano e animais. Estrat?gias de manejo destes res?duos, como a compostagem, s?o importantes ferramentas para garantir seguridade de seu uso como fertilizante. Neste contexto, o objetivo deste trabalho foi avaliar a presen?a de genes de resist?ncia a antimicrobianos em solos de cultivos agr?colas tratados com cama de avi?rio fresca e de reserva legal pr?ximas ?s ?reas agr?colas em Nova Friburgo, RJ, a fim de elucidar o papel destes ambientes como reservat?rio e fonte de dissemina??o da resist?ncia a antimicrobianos. Foi tamb?m avaliado, o efeito do processo de compostagem da cama de avi?rio na preval?ncia de genes de resist?ncia. Ap?s a extra??o do DNA total das amostras de solo, foi realizada a detec??o dos genes de resist?ncia a ?-lact?micos, colistina e sulfonamidas (blaampC, mcr-1, sul1 e sul2, respectivamente) pela t?cnica de PCR (Polymerase Chain Reaction), seguida de uma an?lise de correla??o da presen?a dos genes em rela??o aos atributos f?sico-qu?micos dos solos. A abund?ncia relativa do gene mcr-1 foi determinada pela t?cnica de qPCR (Quantitative Polymerase Chain Reaction). A presen?a dos genes de resist?ncia tamb?m foi avaliada nos tempos 0, 30, 60, 90 e 120 dias de compostagem da cama de avi?rio. Houve diferen?a significativa (P < 0,05) entre as propor??es dos genes sul1 e sul2 nas ?reas de cultivo agr?cola e reserva legal, sendo prevalentes nas ?reas de cultivo agr?cola. O gene mcr-1 foi detectado em todas as amostras de solo. O log da abund?ncia relativa do gene mcr-1 variou de -1,76 (1,81 x 10-? c?pias de mcr-1/16S rDNA) a -3,12 (7,67 x 10-4 c?pias de mcr-1/16S rDNA). O gene blaampC n?o foi detectado ap?s 30 dias de compostagem. J? os genes sul1, sul2 e mcr-1 foram detectados at? 120 dias de compostagem. Esses resultados refor?am a import?ncia de estudos que visem elucidar as vias de dissemina??o de genes de resist?ncia a antimicrobianos nas ?reas de produ??o agr?cola, bem como os fatores que interferem na persist?ncia e dissemina??o desses genes no ambiente, a fim de subsidiar a implementa??o de pr?ticas de manejo que diminuam o risco de dissemina??o da resist?ncia, que constitui uma amea?a potencial ? sa?de p?blica.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2021-07-28T04:30:36Z No. of bitstreams: 1 2018 - Nat?lia dos Santos Ferreira.pdf: 1875758 bytes, checksum: be7275c8691964d6061e97c9ad787431 (MD5) Made available in DSpace on 2021-07-28T04:30:36Z (GMT). No. of bitstreams: 1 2018 - Nat?lia dos Santos Ferreira.pdf: 1875758 bytes, checksum: be7275c8691964d6061e97c9ad787431 (MD5) Previous issue date: 2018-08-09 Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES, Brasil) The application of pre-germinated seedlings from mini-toletes is a recent trend in sugarcane production systems. This system together with the inoculation of plant growth promoting microorganisms can be considered sustainable practices to increase the productivity of the sugar cane crop. The present work aimed to characterize the Rhizobium strain (BR 10268) at a phylogenetic level, to evaluate its potential in promoting the process of germination of IR 42 rice and sprouting of sugarcane mini-setts cv.RB867515, to verify the colonization of mini- and investigate possible chemical mechanisms responsible for the growth promoter effect. Phylogenetic analyzes have positioned the strain in a clade with five microsoimbionte species of legumes based on tree topologies of housekeeping genes glnII and recA indicating that the strain is not a new species of Rhizobium. Experiments conducted with inoculation of the rice strain promoted shoot growth when inoculated with the overdose of the strain. Another experiment conducted under axenic conditions with mini-toletes inoculated with the BR 10268 strain evaluated at 3, 6, 8, 10, 12, and 14 days after inoculation (DAI) demonstrated better sprouting when compared to uninoculated, with significant increases (p?0.05) of the aerial part length and increase of the dry mass of the aerial part between the collection days. Subsequently, for characterization of endogenous colonization of strain, repositioning and quantification of fresh tissue samples, with yolk disinfestation in this work and root disinfestation by the work. The results indicate that [(1.8 ? 0.92) * 107 CFU.g-1 at 7 DAI and (4.9 ? 2.7) * 106 CFU.g-1 at 20 DAI] of yolk and roots [ (2.1 ? 0.75) * 105 CFU.g-1 to 7 DAI and (5.6 ? 4.59) * 104 CFU.g-1 to 20 DAI] could be recovered. Analyzes by BOX-PCR confirm the identity of the recovered cells and the undetected strain in the uninoculated control. Endophytic colonization at 3 and 8 DAI was also confirmed by laser scanning confocal microscopy using a derivative of BR 10268 expressing mCherry. After identification of shoot acceleration and high colonization of the strain in cane internal tissue, analyzes using a supernatant solution of the strain in defined medium were performed by gas chromatography mass spectrometry in ionic selective monitoring (GC-MS / SIM). The results indicate the presence of phytohormones in the range: indoletic acid (30,23) cytokinin (3.39) and active and inactive gibberellic acids GA3, GA4, GA7, GA9, GA12 and GA20 (22,52; 33, 45; 20,59; 5,71; 10,11; 3,22 and 13,66, ng.mL-1, the obligatory motif of the present study confirmed by Rhizobium (BR 10268) is an effective endophyte of sugarcane, which stimulates the sprouting of the aerial part of mini-toletes, through the production of vegetable phytohormones that can be used for biotechnological purposes. A aplica??o de mudas pr?-brotadas a partir de mini-toletes ? uma tend?ncia recente nos sistemas de produ??o de cana-de-a??car. Este sistema juntamente com a inocula??o de microrganismos promotores de crescimento vegetal podem ser consideradas pr?ticas sustent?veis para incrementar a produtividade da cultura da cana-de-a??car. O presente trabalho buscou caracterizar a estirpe Rhizobium (BR 10268) em n?vel filogen?tico, avaliar seu potencial em promover o processo de germina??o de arroz IR 42 e brota??o de mini-toletes de cana-de-a??car cv RB867515, verificar a coloniza??o de mini-toletes de cana e ainda investigar poss?veis mecanismos qu?micos respons?veis pelo efeito promotor de crescimento. An?lises filogen?ticas de genes housekeeping glnII e recA posicionaram a estirpe em um clado com cinco esp?cies de Rhizobium. Experimentos conduzidos com inocula??o da estirpe em arroz, promoveram o crescimento da parte a?rea quando inoculado com o sobredandante da estirpe. Outro experimento conduzido sob condi??es ax?nicas com mini-toletes inoculados com a estirpe BR 10268 avaliados aos 3, 6, 8, 10, 12, e 14 dias ap?s inocula??o (DAI) demonstraram melhor brota??o quando comparados ao n?o inoculado, com aumentos significativos (p?0,05) do comprimento da parte a?rea e aumento da massa seca da parte a?rea entre os dias coletas. Posteriormente para caracteriza??o de coloniza??o endof?tica da estirpe, procedimentos de reisolamento e quantifica??o de amostras de tecidos frescos, com desinfesta??o supercial de gema desenvolvida neste trabalho e desinfesta??o de ra?zes por metodologia tradicional foram realizados. Os resultados indicaram que [(1,8 ? 0,92) * 107 UFC.g-1 a 7 DAI e (4,9 ? 2,7) * 106 UFC.g-1 a 20 DAI] de gema e ra?zes [(2,1 ? 0,75) * 105 UFC.g-1 a 7 DAI e (5,6 ? 4,59) * 104 UFC.g-1 a 20 DAI] poderam ser recuperados. An?lises por BOX-PCR confirmaram a identidade das bact?rias recuperadas e a estirpe n?o foi detectada no controle n?o inoculado. A coloniza??o endof?tica em 3 e 8 DAI tamb?m foi confirmada por microscopia confocal de varredura a laser usando um derivado da BR 10268 expressando mCherry. Ap?s identifica??o de aceleramento da brota??o e elevada coloniza??o da estirpe no tecido interno de cana, an?lises utilizando uma solu??o de sobrenadante da estirpe em meio definido, foi realizada atrav?s de cromatografia de g?s espectrometria de massa em monitoramento seletivo i?nico (GC-MS/SIM). Os resultados indicaram a presen?a de fitohorm?nios produzidos pela estirpe: ?cido indol ac?tico (30,23) citocinina (3,39) e ?cidos giber?licos ativos e inativos GA3, GA4, GA7, GA9, GA12 e GA20 (22,52; 33,45; 20,59; 5,71; 10,11; 3,22 e 13,66 ng.mL-1, respectivamente. Portanto, o presente estudo confirma que Rhizobium sp. (BR 10268) ? um end?fito, efetivo da cana-de-a??car, que estimula a brota??o da parte a?rea de mini-toletes, atrav?s da produ??o de fitohorm?nios vegetais que podem ser utilizados para fins biotecnol?gicos.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2021-05-11T01:43:48Z No. of bitstreams: 1 2018 - Carlos Magno dos Santos.pdf: 3104854 bytes, checksum: 068c4ea7ffa3e7251c3ee89b33831f6f (MD5) Made available in DSpace on 2021-05-11T01:43:49Z (GMT). No. of bitstreams: 1 2018 - Carlos Magno dos Santos.pdf: 3104854 bytes, checksum: 068c4ea7ffa3e7251c3ee89b33831f6f (MD5) Previous issue date: 2018-02-28 Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES, Brasil) The endophytic bacterial community is described as the one able to colonize the internal tissues of the plants without causing apparent prejudice, providing for their hosts development benefits and protection against pathogens. Some of these mechanisms remain little understood, mainly due to the low number of endophytic bacterial cells compared to the plant, and difficulties in accessing the genetic content of that community. On sugarcane, studies about this community were focused mainly on the identification of diazotrophs microorganisms. Generally, works that sought to describe this community were based on methodologies that used the isolation of microorganisms for cultivation. Today it is known that, due to the low depth and coverage of this methodology, can limit the access to these microorganisms. The introduction of culture-independent approaches has allowed a broader analysis of this community and contributing to the understanding of the ecological role of these microorganisms in endosphere of their hosts. The present study used two culture-independent approaches to access the bacterial community on sugarcane. The first was developed a methodology for enrichment of endophytic bacterial cells from the stem tissue of sugarcane. This approach was based on a method of physical enrichment and not biased, that helped surpassing the high level of interfering, as polysaccharides, phenolic compounds, nucleases and fibres. The second approach involved the use of apoplast sap from sugarcane stems. This niche has been described as the main site of colonization by endophytes. The found community is rich and diverse, formed by genera belonging to the Proteobacterias group. One of the main Operational Taxonomic Units (OTUs) observed in the analyses were classified as being of the Sphingomonas genus. The diversity of this genus in sugarcane was also evaluated in this study, and possibly, there are new species present. It was also possible to detect the presence of plant growth promoting bacteria, diazotrophic and pathogens-antagonists. Through predictive analysis of possible genes related to the main OTUs detected, was possible find genes of biotechnological interest, applicable in various areas, the main ones being involved to bioremediation and the improvement of plant cultivation. Thus, this study provides information relevant for understanding the endophytic bacterial community of sugarcane and its functions. A comunidade bacteriana endof?tica ? descrita como aquela capaz de colonizar os tecidos internos das plantas sem causar aparente preju?zo, proporcionando a seus hospedeiros benef?cios para o seu desenvolvimento e prote??o contra pat?genos. Alguns desses mecanismos permanecem pouco compreendidos, principalmente devido ao baixo n?mero de c?lulas bacterianas endof?ticas em compara??o com a planta e dificuldades em acessar o conte?do gen?tico dessa comunidade. Na cana-de-a??car os estudos sobre esta comunidade foram focados principalmente na identifica??o de microrganismos diazotr?ficos. De uma forma geral, trabalhos que visaram descrever esta comunidade foram baseados em metodologias que utilizavam o isolamento dos microrganismos por cultivo. Hoje ? sabido que, devido ? baixa profundidade e cobertura desta metodologia, pode limitar o acesso a estes microrganismos. A introdu??o de abordagens independentes de cultivos tem permitido uma an?lise mais ampla desta comunidade e contribuindo para a compreen??o do papel ecol?gico destes microrganismos na endosfera de seus hospedeiros. O presente trabalho utilizou duas abordagens independentes de cultivo para acessar a comunidade bacteriana na cana-de-a??car. A primeira foi desenvolvida uma metodologia de enriquecimento das c?lulas bacterianas endof?ticas a partir dos tecidos de colmo de cana-de-a??car. Esta abordagem foi baseada em um m?todo de enriquecimento f?sico e n?o direcionado, que ajudou a contornar o alto n?vel de interferentes, como polissacar?deos, compostos fen?licos, nucleases e fibras. A segunda abordagem envolveu o uso do l?quido do apoplasto de colmo da cana-de-a??car. Este nicho tem sido descrito como o principal s?tio de coloniza??o pelos end?fitos. A comunidade encontrada ? rica e diversa, sendo formada por g?neros pertencentes ao grupo das Proteobacterias. Uma das principais Unidades Taxon?micas Operacionais (OTUs) observadas nas an?lises foram classificadas como sendo do g?nero Sphingomonas. A diversidade deste g?nero em cana-de-a??car tamb?m foi avaliada neste estudo, e possivelmente h? a presen?a de novas esp?cies. Foi poss?vel detectar tamb?m a presen?a de bact?rias promotoras de crescimento vegetal, diazotr?ficas e pat?genos-antagonistas. Por meio de uma an?lise preditiva dos poss?veis genes relacionados as principais OTUs detectadas foi poss?vel encontrar genes de interesse biotecnol?gico, aplic?veis em diversas ?reas, sendo os principais envolvidos ? biorremedia??o e o melhoramento do cultivo vegetal. Dessa forma, o presente estudo traz informa??es relevantes ao entendimento da comunidade bacteriana endof?tica de cana-de-a??car e suas fun??es.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2021-06-23T12:07:50Z No. of bitstreams: 1 2018 - Greiciane Fran?a Bronzato de Almeida.pdf: 2781462 bytes, checksum: f0cc862a932387aa170377e23958c01d (MD5) Made available in DSpace on 2021-06-23T12:07:50Z (GMT). No. of bitstreams: 1 2018 - Greiciane Fran?a Bronzato de Almeida.pdf: 2781462 bytes, checksum: f0cc862a932387aa170377e23958c01d (MD5) Previous issue date: 2018-02-23 Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES Dairy farming has been of great importance for the world economy and for such reason producers have, more and more, sought improvements in milk quality, focusing on controlling diseases that affect the cattle such as bovine mastitis. Environmental mastitis, which is commonly caused by microorganisms such as Escherichia coli, can generate great impacts on dairy cattle. This bacterial species has genetic heterogeneity and population characterized by genetically diverse strains, besides the capacity to persist in production environment for an extended period. In this context, the present study aims to evaluate the clonal relationships of E. coli in a milk production environment through the molecular typing techniques Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). For this purpose, 444 samples distributed between milk, faeces, water and the production chain from a farm located in the municipality of Barra do Pira?, in the State of Rio de Janeiro, Brazil, were subjected to biochemical tests and theMatrix-Assisted Laser Desorption Ionization?Time of Flight Mass Spectrometry (MALDI-TOF MS) technique for bacterial identification. In addition, water samples (well, pond, drinking fountain, faucet and creek) were subjected to the Most Probable Number (MPN) test for total and thermotolerant coliform evaluation, in which they presented potability standards higher than those stipulated by the Ministry of Health. Out of 183 identified enterobacteria, 152 (83%) were confirmed by both methodologies as being E. coli. Out of these, nine representatives had the gyrB gene sequenced for molecular confirmation of the species exhibiting up to 99% maximum identity when the sequences were compared to the NCBI database sequences. Later, all 152 E. coli strains were phenotypically subjected to biofilm production and virulence genes detection, where it was possible to observe that 41.44% (63/152) of strains were biofilm producers, being 37.5% (57/152) weak producers, 1.31% (2/152) of moderate producers and 2.63% (4/152) strong producers. Moreover, 92.1% (140/152) of fimH gene positive strains could be detected, 88.8% (135/152) for the csgA gene, 29.6% (45/152) for the flu gene (which are biofilm-related genes) and 13.1% (20/152) positive for the eaeA gene; 7.2% (11/152) for the LT gene and 2.6% (4/152) for the stxI gene. No strain was positive for the stxII, ST, ial and eagg genes. From these results, 18 profiles were established with the purpose of selecting 30 strains that were processed in the Laboratory of Genetics of the National University of R?o Cuarto, Argentina, through the PFGE technique, where it was possible to observe high genetic variability. Strains that presented from 95% to 100% of similarity (n = 10) were sequenced in order to establish a clonal relationship between them through the MLST technique that generated eight different ST types, with ST164 and ST1308 being the ones that established possible clonal relationships between strains. Furthermore, a new type of sequence (ST) was observed, one which should be subjected to a new generation sequencing and then sent to the MLST healer to generate a new ST number and deposited in the schema database. When researching the database as well as the literature, no ST reports (ST5, ST164, ST165 and ST1308) derived from bovine E. coli strains were found in Brazil, leading to the understanding that this work is the very first report in such country. A atividade leiteira tem sido de grande import?ncia para economia em todo o mundo e, por esse motivo, cada vez mais os produtores t?m buscado melhorias na qualidade do leite, focando no controle de enfermidades que acometem o rebanho, em especial da mastite bovina. A mastite ambiental pode gerar grandes impactos na bovinocultura leiteira, sendo esta comumente ocasionada por microrganismos como Escherichia coli. Esta esp?cie bacteriana possui heterogeneidade gen?tica e popula??o caracterizada por cepas geneticamente diversificadas, al?m da capacidade de persistir no ambiente de produ??o por tempo prolongado. Frente a esse contexto, o presente estudo teve como objetivo avaliar as rela??es clonais de E. coli em ambiente de produ??o leiteira atrav?s das t?cnicas de tipagem molecular Pulsed Field Gel Eletrophoresis (PFGE) e Multilocus Sequence Typing (MLST), para isso 444 amostras distribu?das entre leite, fezes, ?gua e cadeia produtiva oriundas de uma fazenda localizada no munic?pio de Barra do Pira? no Estado do Rio de Janeiro, Brasil, foram submetidas a testes bioqu?micos e a t?cnica de MALDI-TOF MS para identifica??o bacteriana. Al?m disso, as amostras de ?gua (po?o, a?ude, bebedouro, torneira e riacho) foram submetidas ao teste do N?mero Mais Prov?vel (NMP) para avalia??o de coliforme totais e termotolerantes no qual apresentaram padr?es de potabilidade superiores aos estipulados pelo Minist?rio da Sa?de. De 183 enterobact?rias identificadas, 152 (83%) foram confirmadas atrav?s de ambas as metodologias como E. coli. Destas, nove representantes tiveram o gene gyrB sequenciado para confirma??o molecular da esp?cie apresentando at? 99% de m?xima identidade quando as sequ?ncias foram comparadas com as sequ?ncias do banco de dados NCBI. Posteriormente, todas as 152 cepas de E. coli foram submetidas fenotipicamente a produ??o de biofilme e detec??o de genes de virul?ncia, onde foi poss?vel observar que 41,44% (63/152) de cepas foram produtoras de biofilme, sendo 37,5% (57/152) fraca produdoras, 1,31% (2/152) produtoras moderadas e 2,63% (4/152) fortes produtoras. Al?m disso, foi poss?vel detectar 92, 1% (140/152) de cepas positivas para o gene fimH; 88,8% (135/152) para o gene csgA, 29,6% (45/152) para o gene flu (que s?o genes relacionados ao biofilme) e 13,1% (20/152) positivas para o gene eaeA; 7,2% (11/152) para o gene LT e 2,6% (4/152) para o gene stxI. Nenhuma cepa foi positiva para os genes stxII, ST, ial e eagg. A partir destes resultados, 18 perfis foram estabelecidos com o prop?sito de selecionar 30 cepas que foram processadas no laborat?rio de gen?tica da Universidad Nacional de R?o Cuarto, Argentina, atrav?s da t?cnica PFGE, onde foi poss?vel observar uma elevada variabilidade gen?tica. As cepas que apresentaram de 95% a 100% de similaridade (n=10) foram sequenciadas, a fim de estabelecer uma rela??o clonal entre elas atrav?s da t?cnica de MLST, que gerou oito tipos diferentes de ST, sendo o ST164 e o ST1308 os que estabeleceram poss?veis rela??es clonais entre cepas. Al?m disso, foi observado um novo tipo de sequ?ncia (ST) que dever? ser submetido a um sequenciamento de nova gera??o, e ent?o ser enviado ao curador do MLST para que seja gerado um novo n?mero de ST e depositado no banco de dados do esquema. Ao realizar buscas tanto no banco de dados quanto na literatura, n?o foram encontrados no Brasil relatos sobre os STs (ST5, ST164, ST165 e ST1308) estudados provenientes de cepas de E. coli bovinas, levando ao entendimento de que este trabalho ? o primeiro relato no pa?s.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2021-05-13T22:16:59Z No. of bitstreams: 1 2018- Carlos Vergara Torres J?nior.pdf: 4875221 bytes, checksum: 8b28f2ec44a9c94d03d3c4e72c56df4f (MD5) Made available in DSpace on 2021-05-13T22:16:59Z (GMT). No. of bitstreams: 1 2018- Carlos Vergara Torres J?nior.pdf: 4875221 bytes, checksum: 8b28f2ec44a9c94d03d3c4e72c56df4f (MD5) Previous issue date: 2018-02-20 Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES, Brasil) Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brasil) A plant colonized by dark septate endophytic (DSE fungi) fungi can better recovery the nutrients provided to it when mutualistic interaction is established, generating financial savings to the farmer. However, several ecological functions of DSE fungi still need to be clarified. The present study aimed to evaluate the effect of dark septate endophytic fungi on the nitrogen recovery efficiency in tomato and rice plants and on the expression of plasma membrane H+-ATPase (PM H+-ATPase) isoforms and nitrate (NO3-) transporters in rice plants. The experiments with tomato and rice were conducted in non-sterile greenhouse soil in a randomized complete block design with five replicates. The treatments consisted of tomato (Santa Clara I-5300 variety) or rice (Piau? variety) plants without (control) and inoculated with DSE fungi A101, A104 and A105, and A101 and A103, respectively. Pots containing non-sterile material from the surface horizon of a Fragiudult soil, received ammonium sulfate enriched with 1 atom % 15N excess or finely ground Canavalia ensiformis [L.] enriched with 2.15 or 0.7 atom % 15N excess as the sole source of N. Growth plant indicators, accumulation of plant dry matter and nutrients, and recovery efficiency of 15N were evaluated at 54 days after transplanting and at maturity stage of rice plants, and at 50 days after transplanting of tomato seedlings. Expression of NO3- transporters and PM H+ -ATPase isoforms and H+ pumps activity was also evaluated in rice plants inoculated with A103. Compared with control, rice and tomato plants were more proficient in the recovery of nutrients, especially N derived from C. ensifomis. Rice plants also responded positively to the inoculation under ammonium sulfate supplementation, accumulating more dry matter and nutrients in relation to the control, and when rice plants were inoculated with the A103 fungus in a sterile environment, activity of PM H+-ATPase and of vacuolar H+-pyrophosphatase, and transcripts abundance of PM H+-ATPase isoforms OsA5 and OsA8 increased. A101, A104 and A105, and A101 and A103 fungi are beneficial for tomato and rice, respectively. Uma planta colonizada por fungos endof?ticos dark septate (fungos DSE), pode aproveitar melhor os nutrientes a ela fornecidos, quando a intera??o mutualista ? estabelecida, gerando economia financeira ao produtor. Entretanto, uma s?rie de fun??es ecol?gicas dos fungos DSE, ainda precisam ser esclarecidas. Este trabalho teve como objetivo, avaliar o efeito de fungos endof?ticos dark septate sobre a efici?ncia de aproveitamento de fontes nitrogenadas em tomate e arroz, e sobre a express?o das isoformas de H+-ATPase da membrana plasm?tica (PM H+-ATPase) e transportadores de nitrato (NO3-) em plantas de arroz. Os experimentos para estudar o efeito dos fungos DSE, na recupera??o pelas plantas do arroz e tomate do N derivado de diferentes fontes, foram conduzidos em solo n?o esterilizado, em casa de vegeta??o, em delineamento de blocos ao acaso, com cinco repeti??es. Os tratamentos consistiram de plantas de tomate (cv Santa Clara I-5300) inoculadas com os fungos DSE A101, A104 ou A105 ou de plantas de arroz (variedade Piau?) inoculadas com os fungos DSE A101 ou A103 e de plantas deixadas sem inocula??o (controle). Vasos contendo amostra de horizonte superficial de um Planossolo H?plico n?o esterilizado, receberam sulfato de am?nio marcado com 1% de ?tomos de 15N em excesso ou Canavalia ensiformis [L.] finamente triturada enriquecida com 2,15% ou 0,7 ?tomos de 15N em excesso, como ?nica fonte de N. Indicadores de crescimento, ac?mulo de mat?ria seca e nutrientes e a efici?ncia de recupera??o de 15N foram avaliados aos 54 dias ap?s o transplantio e no est?dio de matura??o do arroz (130 dias ap?s o transplantio) e aos 50 dias ap?s o transplantio de pl?ntulas de tomate. A express?o de transportadores de NO3- e isoformas PM H+-ATPase e atividade de bombas de H+ foram tamb?m avaliados em plantas de arroz inoculadas com A103, em condi??es esterilizadas. Em compara??o com o controle, as plantas de arroz e tomate foram mais eficientes no aproveitamento de nutrientes, em especial do N derivado da C. ensifomis. As plantas de arroz tamb?m responderam positivamente ? inocula??o, sob suplementa??o com sulfato de am?nio, acumulando mais mat?ria seca e nutrientes em rela??o ao controle, e quando foram inoculadas com o fungo A103 em ambiente esterilizado, aumentaram a atividade da PM H+-ATPase e da H+-pirofosfatase vacuolar e abund?ncia de transcritos das isoformas de PM H+-ATPase OsA5 e OsA8. Os fungos A101, A104 e A105 s?o ben?ficos para o tomate e os fungos A101 e A103 para o arroz.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2019-03-20T18:21:10Z No. of bitstreams: 1 2017 - Ivan de Alencar Menezes J?nior.pdf: 2048768 bytes, checksum: 10fd425fb0df338a0e4159e5c6a4d1ba (MD5) Made available in DSpace on 2019-03-20T18:21:10Z (GMT). No. of bitstreams: 1 2017 - Ivan de Alencar Menezes J?nior.pdf: 2048768 bytes, checksum: 10fd425fb0df338a0e4159e5c6a4d1ba (MD5) Previous issue date: 2017-04-27 CAPES The genus Bradyrhizobium is commonly found as endosymbiont in legumes (Fabaceae family), such as soybean (Glycine max), forming nodules on the roots and providing nitrogen from the atmosphere to these plants and to the soil. Recent research has shown the presence of bacteria from this genus inside roots of sugarcane (Saccharum spp.), a non-legume belonging to the Poaceae family. This work aims to know the population density of these bacteria associated with the roots of two sugarcane cultivars and to understand how the plant genotype (cultivar) affects the population composition of these bacteria found in the rhizosphere and the endosphere. Rhizosphere and root samples of two economically relevant sugarcane cultivars (RB867515 and IACSP95-5000) were collected in the experimental field of Embrapa Agrobiology, serially diluted and inoculated on gnotobiotic seedlings of cowpea used as trap plants. The most probable number technique was used to quantify the density of the rhizobia present in the samples and the isolates obtained from the nodules were cultivated to form pure cultures. BOX-PCR technique and sequence analysis of the ribosomal internally transcribed spacer region (ITS), recA housekeeping gene, and the symbiotic nodC gene were applied to better understand the genetic diversity of these rhizobia. Acetylene reduction test was applied to verify the presence of nitrogenase activity under free-living conditions. The presence of at least 15625 rhizobia g root -1 in rhizosphere samples and between 125 and 625 rhizobia g root-1 in the endosphere samples was observed. This shows that the density of rhizobia in the rhizosphere of sugarcane is significantly higher than in the interior of the roots for the two cane cultivars. A total of 34 isolates were obtained from the rhizosphere samples of each cultivar and 15 and 14 respectively from the roots of cultivars IACSP95-5000 and RB867515. Most of the isolates presented morphoculture characteristics of the genus Bradyrhizobium. Phylogenetic analyzes positioned most of the isolates in the superclade Bradyrhizobium elkanii. No correlation was observed between the plant cultivar and the abundance of Bradyrhizobium spp. in the roots and rhizosphere of sugarcane plants. O g?nero Bradyrhizobium ? comumente encontrado como endossimbionte em leguminosas (fam?lia Fabaceae) como a soja (Glycine max), formando n?dulos nas ra?zes dessas plantas e disponibilizando nitrog?nio da atmosfera para essas plantas e para o solo. Pesquisas recentes mostraram a presen?a de bact?rias desse g?nero no interior de ra?zes de cana-de-a??car (Saccharum spp.), uma n?o leguminosa pertencente ? fam?lia Poaceae. Este trabalho tem como objetivo conhecer a densidade populacional dessas bact?rias associadas ?s ra?zes de duas cultivares de cana-de-a??car e compreender como o gen?tipo da planta (cultivar) afeta a composi??o populacional dessas bact?rias encontradas na rizosfera e na endosfera. Amostras de rizosfera e de ra?zes de duas cultivares econ?micamente relevantes de cana-de-a??car (RB867515 e IACSP95-5000) foram coletadas no campo experimental da Embrapa Agrobiologia, seriadamente dilu?das e inoculadas em pl?ntulas gnotobi?ticas de feij?o-caupi usadas como plantas iscas. A t?cnica de n?mero mais prov?vel foi utilizada para quantificar a densidade dos riz?bios presentes nas amostras e os isolados obtidos dos n?dulos foram cultivados at? formarem culturas puras. A t?cnica de BOX-PCR e an?lise de sequ?ncia dos genes recA, regi?o ITS e nodC foram aplicadas para compreender melhor a diversidade gen?tica desses riz?bios. Ensaio de redu??o de acetileno foi aplicado para verificar a presen?a de atividade da nitrogenase. Observou-se a presen?a de no m?nimo 15625 riz?bios g -1 de raiz em amostras de rizosfera e entre 125 e 625 riz?bios g -1 de raiz nas amostras de endosfera. Isso mostra que a densidade de riz?bios na rizosfera de cana-de-a??car ? significantemente maior do que no interior das ra?zes para as duas cultivares de cana. Foram obtidos 34 isolados das amostras de rizosfera de cada cultivar e 15 e 14 respectivamente do interior das ra?zes das cultivares IACSP95-5000 e RB867515. A maior parte dos isolados apresentaram caracter?sticas morfoculturais do g?nero Bradyrhizobium. An?lises filogen?ticas posicionaram a maioria dos isolados no superclado de Bradyrhizobium elkanii. N?o foi observada correla??o entre a cultivar vegetal e a abund?ncia de Bradyrhizobium. spp. nas ra?zes e rizosfera das plantas de cana-de-a??car.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2018-09-13T17:03:37Z No. of bitstreams: 1 2017 - Gustavo Feitosa de Matos.pdf: 1844017 bytes, checksum: 0ed6966082e21d15123a12cd0e5777ef (MD5) Made available in DSpace on 2018-09-13T17:03:38Z (GMT). No. of bitstreams: 1 2017 - Gustavo Feitosa de Matos.pdf: 1844017 bytes, checksum: 0ed6966082e21d15123a12cd0e5777ef (MD5) Previous issue date: 2017-02-23 CNPq - Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico Recent molecular biology studies have indicated that bacteria of the genus Bradyrhizobium and Rhizobium may also play a role in the biological nitrogen fixation process (BNF) when associated with sugarcane. The use of Vigna unguiculata trap plants allowed obtaining a diverse collection of endophytic Bradyrhizobium spp. from sugarcane roots. The present work aimed to characterize a group of bacteria of the genus Bradyrhizobium from sugarcane roots, as well as to evaluate the ability of a representative strain of this group and an isolate of the genus Rhizobium to promote growth in this crop. To define the taxonomic position of the isolates of the group, phylogenetic analyzes were performed with the individual 16S rRNA, ITS, nodC and recA genes and multilocus sequence analysis (MLSA) involving four housekeeping genes (recA, dnaK, glnII and atpD), in four representative isolates (BR 10280T, BR 10266, BR 10555 and BR 10556). In addition, biochemical and morphophysiological tests were performed. Experiments in which the isolates P9-20 (BR 10280) (Bradyrhizobium sp.) and P5-2 (Rhizobium sp.) were inoculated in sugarcane seedlings were conducted in a greenhouse to evaluate the growth promoting effect of these isolates. Two harvests were performed at 30 and 75 days after transplanting (DAT). Among the analyzed variables were the budding speed index (BSI), dry mass and total nitrogen (N). Phylogenetic analyzes positioned the isolates in the superclade of B. japonicum in an independent branch close to B. huanghuaihaiense, a soybean micro-symbiont. Despite the proximity of the group under study to B. huanghuaihaiense, these isolates did not induce nodulation in Glycine max. In addition, unlike B. huanghuaihaiense, the new isolates induced nodule formation in Phaseolus vulgaris. In morphophysiological studies, significant differences were demonstrated between the representative isolates from this study and B. huanghuaihaiense. In the greenhouse experiment, an increase on BSI and root dry mass of the inoculated treatment with the P5-2 isolate was observed in the first harvest. In the second harvest, increments were found in the root dry mass and root volume, as well as in the total N of the roots and aerial part, in the treatment inoculated with P5-2, although no statistical difference was detected. These results of the characterization indicate that the sugarcane isolates of the genus Bradyrhizobium represent a new species of this genus. In relation to the greenhouse experiment, the isolate of the genus Rhizobium sp. presented potential as a growth promoter in the sugarcane crop. Estudos recentes de biologia molecular indicaram que bact?rias do g?nero Bradyrhizobium e Rhizobium tamb?m podem ter um papel no processo de fixa??o biol?gica de nitrog?nio (FBN) quando associadas ? cana-de-a??car. O uso de plantas de Vigna unguiculata como ?isca? possibilitou a obten??o de uma cole??o diversa de Bradyrhizobium spp. endof?ticos a partir de ra?zes de cana-de-a??car. O presente trabalho teve como objetivo caracterizar um grupo de bact?rias do g?nero Bradyrhizobium proveniente de ra?zes de cana-de-a??car, assim como avaliar a capacidade de uma estirpe representante desse grupo e de um isolado do g?nero Rhizobium em promover efeito de crescimento nessa cultura. Para definir o posicionamento taxon?mico dos isolados do grupo, an?lises filogen?ticas foram realizadas com os genes individuais 16S rRNA, ITS, nodC e recA e an?lise de sequ?ncia de multilocus (MLSA) envolvendo quatro genes ?housekeeping? (recA, dnaK, glnII e atpD), em quatro isolados representantes (BR 10280T, BR 10266, BR10555 e BR 10556). Al?m disso, foram realizados testes bioqu?micos e morfofisiol?gicos. Experimento onde os isolados P9-20 (BR 10280) (Bradyrhizobium sp.) e P5-2 (Rhizobium sp.) foram inoculados em minitoletes de cana-de-a??car foi conduzido em casa de vegeta??o. Foram realizadas coletas aos 30 e 75 dias ap?s transplantio (DAT). Entre as vari?veis analisadas est?o o ?ndice de velocidade de brotamento (IVB), massa seca e Nitrog?nio (N) total. An?lises filogen?ticas posicionaram os isolados no super clado de B. japonicum em um ramo independente pr?ximo a B. huanghuaihaiense, um microssimbionte de soja. Apesar da proximidade do grupo em estudo com B. huanghuaihaiense, estes isolados n?o induziram nodula??o em Glycine max. Al?m disso, diferente de B. huanghuaihaiense, os novos isolados induziram a forma??o de n?dulos em Phaseolus vulgaris. Em estudos morfofisiol?gicos foram demostradas diferen?as significativas entre os isolados representantes do grupo em estudo e B. huanghuaihaiense. Em experimento em casa de vegeta??o, observou-se na primeira coleta um incremento sobre o IVB e massa seca da raiz no tratamento inoculado com o isolado P5-2, embora n?o tenham sido observadas diferen?as estat?sticas. Na segunda coleta, incrementos foram encontrados na massa seca de raiz e volume de raiz, assim como, no N total das ra?zes e parte a?rea, no tratamento incoculado com P5-2, embora n?o tenham apresentado diferen?a estat?stica. Massa seca da parte a?rea do tratamento inoculado com P5-2 apresentou incremento na primeira e segunda coleta, em rela??o aos controles, embora n?o tenha sido detectada diferen?a estat?stica. Os resultados da caracteriza??o do grupo indicam que os isolados de cana-de-a??car do g?nero Bradyrhizobium representam uma nova esp?cie desse g?nero. Em rela??o ao experimento em casa de vegeta??o, o isolado do g?nero Rhizobium sp. apresentou potencial como promotor de crescimento na cultura de cana-de-a??car

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-08-23T18:13:31Z No. of bitstreams: 1 2015 - Jakson Leite.pdf: 1055808 bytes, checksum: fa105f74410a81a1f30e45ae5e911c9d (MD5) Made available in DSpace on 2017-08-23T18:13:31Z (GMT). No. of bitstreams: 1 2015 - Jakson Leite.pdf: 1055808 bytes, checksum: fa105f74410a81a1f30e45ae5e911c9d (MD5) Previous issue date: 2015-02-27 CAPES Cowpea [Vigna unguiculata (L.) Walp] is an important crop in northeastern Brazil with strategic advantages for production in semi-arid region, such its drought tolerance and good performance in low fertility soils. In addition, the nitrogen (N) fixed in symbiosis with rhizobia eliminates the demand for N fertilizers, with economic, social and environmental benefits. Little is known about the genetic diversity of bacteria associated to cowpea nodules in Brazilian semi-arid. The aim of the study was to characterize the bacterial diversity of Brazilian semi-arid soils associated with nodules of different cowpea cultivars by dependent and independent bacterial cultivation strategy. Initially a collection of 86 bacteria cowpea nodules isolated from semiarid soils was genetically characterized by partial 16S rRNA gene sequencing and symbiotic genes nifH and nodC. The sequences were compared with the NCBI database to identify isolates and phylogenetic relationships were built. In another study, we applied the independent cultivation method to evaluate bacterial communities associated with the nodules of two cowpea cultivars (BRS and BRS Acau? Pujante), in Ultisol with no history of cowpea cultivation. Nodules (N) were collected 35 days after germination, and soil samples (BS) from 0-20 cm deeper. DNA was extracted for analysis of bacterial communities with 454 pyrosequencing of the 16S ribosomal gene rRNA. The analysis of the diversity of the bacterial collection of the nodules 54 of the 86 isolates were Bradyrhizobium. Other (32) belong to Rhizobium (13) and Microvirga (1), Alfaproteobact?ria class; Burkholderia (8), and Ralstonia (1), Betaproteobacteria class; Acinetobacter (1), Cronobacter (3), Enterobacter (1), and Pantoea (1), Gamaproteobact?ria; and Leifsonia (3), phylum Actinobacteria. As Bradyrhizobium predominated, analyzes were performed with the almost full 16S rRNA, nifH and nodC and isolates were distributed in 5 lines: 16S rRNA type I (44 isolates), type II (6), Type III (1), Type IV ( 2) and type IV (1). Phylogenetic analysis of the 16S rRNA gene grouped the Type I strain in the large group Bradyrhizobium japonicum and close to the type strain of Bradyrhizobium yuanmingense. The analyses of the nifH and nodC gene separated the isolates in 5 symbiotic lines (I, II, III, IV and IV) and were congruent among them, which supports the theory of monophyletic in origin symbiotic gene Bradyhrizobium. The symbiotic lineages I and II are nearby and correspond to all isolates with 16S rRNA type I, being the dominant group associated with nodules. The partial 16S rRNA gene sequencing of bacterial communities showed high diversity in the three environments (BS, RS and N). The communities associated with the nodes were significantly different (p> 0.01) from the surrounding nodules (LS and RS). Phyla Actinobacteria, Bacteriodetes, Proteobacteria were plentiful for BS and RS. In nodes, the Proteobacteria and Bacteriodetes phyla predominated, Gammaproteobacteria being (58.8%) and Alphaproteobacteria (37.4%) in the phylum Proteobacteria and dominant Flavobacteriia (84.8%) and Sphingobacteriia (10.9%) in the phylum Bacteriodetes. For gender, Chryseobacterium, Entreobacter and Bradyrhizobium dominate in all nodes samples where Chryseobacterium prevailed in BRS Acau? and Enterobacter in BRS Pujante. O feij?o-caupi [Vigna unguiculata (L.) Walp] ? uma das principais culturas no Nordeste do Brasil com vantagens estrat?gicas para produ??o no semi?rido, como toler?ncia a seca e bom desempenho em solos de baixa fertilidade. Al?m disso, fixa N em simbiose com riz?bios eliminando a demanda de fertilizantes nitrogenados, com benef?cios econ?micos, sociais e ambientais. Pouco se sabe sobre a diversidade gen?tica de bact?rias associadas aos n?dulos de feij?o-caupi no semi?rido. O objetivo do estudo foi caracterizar a diversidade de bact?rias de solos do semi?rido brasileiro associadas aos n?dulos de diferentes cultivares de feij?o-caupi com arbordagem que depende e independe de cultivo das bact?rias. Inicialmente uma cole??o de 86 bact?rias de n?dulos de feij?o-caupi isoladas de solos do semi?rido foi caracterizada geneticamente pelo sequenciamento parcial do gene 16S rRNA e dos genes simbi?ticos nifH e nodC. As sequ?ncias foram comparadas com as do banco de dados do NCBI para identificar os isolados e as rela??es filogen?ticas dos mesmos com as de esp?cies conhecidas. Em outro estudo, aplicou-se o m?todo independente de cultivo para avaliar comunidades de bact?rias associadas aos n?dulos de dois cultivares de feij?o-caupi (BRS Pujante e BRS Acau?), em Argissolo Amarelo sem hist?rico de uso com a lavoura. Os n?dulos (N) foram coletados 35 dias ap?s a germina??o e a amostragem do solo (BS) de 0-20 cm. O DNA das amostras foi extra?do para an?lises das comunidades bacterianas com 454 pirosequenciamento do gene ribossomal 16S rRNA. Na an?lise da diversidade da cole??o de n?dulos 54 dos 86 dos isolados foram de Bradyrhizobium. Os demais (32) pertencem aos g?neros Rhizobium (13) e Microvirga (1), classe Alfaproteobact?ria; Burkholderia (8) e Ralstonia (1), classe Betaproteobact?ria; Acinetobacter (1), Cronobacter (3), Enterobacter (1) e Pantoea (1), Gamaproteobact?ria; e Leifsonia (3), filo Actinobact?ria. Como Bradyrhizobium predominou, foram feitas an?lises com os genes 16S rRNA, nifH e nodC e os isolados distribu?ram-se em 5 linhagens: 16S rRNA tipo I (44 isolados), tipo II (6), tipo III (1), tipo IV (2) e tipo IV (1). A an?lise filogen?tica do gene 16S rRNA agrupou a linhagem tipo I no grande grupo Bradyrhizobium japonicum e pr?ximo da estirpe tipo de Bradyrhizobium yuanmingense. A an?lise dos genes nifH e nodC separou os isolados em 5 linhagens simbi?ticas (I, II, III, IV e IV) e as ?rvores foram congruentes, o que suporta a teoria da origem monofil?tica de genes simbi?ticos em Bradyhrizobium. As linhagens simbi?ticas I e II s?o pr?ximas e correspondem a todos os isolados com 16S rRNA tipo I, sendo o grupo dominante associado aos nodulos. O sequenciamento parcial do gene 16S rRNA das comunidades bacterianas mostrou alta diversidade nos tr?s ambientes (BS, RS e N). As comunidades associadas aos n?dulos foram significativamente diferentes (p> 0,01) das que cercam os n?dulos (LS e RS). Os filos Actinobacteria, Bacteriodetes, Proteobacteria foram abundantes para BS e RS. Em n?dulos, os filos Proteobacteria e Bacteriodetes predominaram, sendo Gammaproteobacteria (58,8%) e Alphaproteobacteria (37,4%) dominantes no filo Proteobacteria e Flavobacteriia (84,8%) e Sphingobacteriia (10,9%) no filo Bacteriodetes. Para g?nero, Chryseobacterium, Entreobacter e Bradyrhizobium dominam em todas as amostras de n?dulos, onde Chryseobacterium predominou em BRS Acau? e Enterobacter em BRS Pujante.