Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-11Bitstream added on 2014-06-13T19:35:20Z : No. of bitstreams: 1 silva_n_me_jabo.pdf: 388385 bytes, checksum: d63f1e8443ba711c0afd71f2f0445af1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Como alternativa ao controle químico, principal método de controle de pragas agrícolas, a bactéria entomopatogênica Bacillus thuringiensis (Bt) é um agente de controle com características tóxicas e ambientais que permitem o controle de insetos-praga de acordo com as premissas do MIP. Com o objetivo de buscar novas linhagens potencialmente tóxicas para Sitophilus oryzae (L., 1763) (Coleóptera, Curculinidae), caracterizou-se molecularmente 1.073 isolados de B. thuringiensis de diferentes partes do Brasil quanto ao conteúdo do gene cry35Ba. O material genético foi extraído através do kit InstaGene Matrix, utilizado para a amplificação das seqüências através da técnica de PCR, sendo os resultados visualizados em gel de agarose 1,5%. A classe do gene cry35Ba foi representada por 60 isolados (5,6%) de Bt, os quais foram submetidos ao bioensaio com larvas de S. oryzae. Dentre os isolados estudados, quatro causaram mortalidade acima de 50% nos testes de patogenicidade, e os isolados 544 e 622 foram os mais virulentos, conforme determinado pela estimativa da CL50. Nos quatro isolados que demonstraram toxicidade, foi detectado através da microscopia eletrônica a presença de cristais esféricos, e um isolado apresentou cristais bipiramidais e cubóides e pôde-se verificar proteínas com 44 kDa, referentes aos genes cry35Ba por SDS-Page. Esses dados demonstram o potencial de Bt no manejo de S. oryzae e estudos são necessários para determinar a melhor forma de utilização desta tecnologia no manejo dessa praga. / An alternative to chemical control, which is the major agricultural pest control method, the entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the IPM precepts. In order to find new strains, potentially toxic to Sitophilus oryzae Lineu, 1763 (Coleoptera: Curculinidae), 1073 strains of B. thuringiensis from different parts of Brazil were characterized molecularly for the cry35Ba gene. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in PCR, and viewed in 1,5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%), which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in the pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL50 estimates. The four isolates the were toxic showed in the electron microscopy the presence of spherical, and isolates one presented crystals bipyramidal and cuboid crystals, and a 44-kDa protein was found in SDS-Page, which correspond to the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae. More studies are required to determine the best use of these isolates for pest management.

Resumo: Como alternativa ao controle químico, principal método de controle de pragas agrícolas, a bactéria entomopatogênica Bacillus thuringiensis (Bt) é um agente de controle com características tóxicas e ambientais que permitem o controle de insetos-praga de acordo com as premissas do MIP. Com o objetivo de buscar novas linhagens potencialmente tóxicas para Sitophilus oryzae (L., 1763) (Coleóptera, Curculinidae), caracterizou-se molecularmente 1.073 isolados de B. thuringiensis de diferentes partes do Brasil quanto ao conteúdo do gene cry35Ba. O material genético foi extraído através do kit InstaGene Matrix, utilizado para a amplificação das seqüências através da técnica de PCR, sendo os resultados visualizados em gel de agarose 1,5%. A classe do gene cry35Ba foi representada por 60 isolados (5,6%) de Bt, os quais foram submetidos ao bioensaio com larvas de S. oryzae. Dentre os isolados estudados, quatro causaram mortalidade acima de 50% nos testes de patogenicidade, e os isolados 544 e 622 foram os mais virulentos, conforme determinado pela estimativa da CL50. Nos quatro isolados que demonstraram toxicidade, foi detectado através da microscopia eletrônica a presença de cristais esféricos, e um isolado apresentou cristais bipiramidais e cubóides e pôde-se verificar proteínas com 44 kDa, referentes aos genes cry35Ba por SDS-Page. Esses dados demonstram o potencial de Bt no manejo de S. oryzae e estudos são necessários para determinar a melhor forma de utilização desta tecnologia no manejo dessa praga. / Abstract: An alternative to chemical control, which is the major agricultural pest control method, the entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the IPM precepts. In order to find new strains, potentially toxic to Sitophilus oryzae Lineu, 1763 (Coleoptera: Curculinidae), 1073 strains of B. thuringiensis from different parts of Brazil were characterized molecularly for the cry35Ba gene. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in PCR, and viewed in 1,5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%), which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in the pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL50 estimates. The four isolates the were toxic showed in the electron microscopy the presence of spherical, and isolates one presented crystals bipyramidal and cuboid crystals, and a 44-kDa protein was found in SDS-Page, which correspond to the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae. More studies are required to determine the best use of these isolates for pest management. / Orientador: Manoel Victor Franco Lemos / Coorientador: Ricardo Antonio Polanczyk / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Uderlei Donisete Silveira Covissi / Mestre

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

Organisms belonging to the Kingdom Fungi are known to occupy a wide variety of ecological niches and are found globally in virtually all environments. Two members of the smallest of the fungal phylum, the Zygomycota, have also been found to harbor intercellular bacteria initially described as being from or closely related to organisms from the Genus Burkholderia. In this study two microaerophilic members of the species Verticilium from the phyla Ascomycota were characterized. Both appear to carry two bacterial endosymbionts. This is the first evidence of bacterial endosymbionts found within a member of the Ascomycota. Through the use of fluorescent stains, isolation of the intercellular bacteria, DNA analysis and fluorescent in situ hybridization (FISH) it appears that the newly isolated Verticilium sp. fungi contain not one but two bacterial endosymbionts from the family Proteobacteria. One putative symbiont is from the genus Bradyrhizobium, a member of the α-Proteobacteria, and one from the genus Burkholderia, a member of the β-Proteobacteria. This is the first evidence of a fungus containing not one, but two distinct endosymbionts from two separate bacterial families. Additionally the fungi were found to grow from spore across a large pH gradient (pH 1.2 to pH 13.5) and in conditions lacking given nutrient. They were tolerant of concentrations of Fe(II) up to 50mM and grew better with low oxygen levels (1.6%) than without.

‘Candidatus Liberibacter solanacearum’ is a α-Proteo bacteria, Gram-negative, restricted to plant phloem and to the haemolymph of psyllids that act as vectors. This emerging bacterium has been associated with different diseases in different hosts and associated with carrot (Daucus carota) in Spain. Vegetative disorders of unknown etiology have also been observed in celery (Apium graveolens) since 2008. A real-time PCR protocol specific to ‘Ca. L. solanacearum’ detection, using TaqMan probe and direct sample preparation methods have been developed. This technology has been validated in an intra-laboratory study (sensitivity 1, specificity 1 and accuracy 100%) and is available commercially as a complete kit. It has been demonstrated that ‘Ca. L. solanacearum’ is associated with the observed syndrome in celery and a new bacterium haplotype (E) has been identified. With these results it is concluded that celery is a new host of ‘Ca. L. solanacearum’ (Teresani et al., 2014a). Using the newly developed real-time PCR protocol ‘Ca. L. solanacearum’ has been detected in 42,6% of the carrot seeds lots tested and in individual seeds. The number of cells/seeds has been estimated in 4.8±3.3 to 210±6.7, which only 5% were viable. After 150 days post-germination, 12% of seedlings showed symptoms and tested positive for ‘Ca. L. solanacearum’. Liberibacter-like cells were observed in the phloem sieve elements of the seed coat and in the phloem of carrot leaf midrib from seedlings. These results demonstrated that ‘Ca. L. solanacearum’ is transmitted by carrot seeds (Bertolini et al., 2014b). The collected arthropods were classified into families, and the superfamily Psylloidea was identified to the species level resulting Bactericera trigonica, B. tremblayi and B. nigricornis the main identified species. The population dynamics of different psyllids species visiting carrot, celery and potato has been determined, concluding that the highest populations are captured during summer. The bacterium has been detected in the different Bactericera species previous cited additionally to Bactericera sp. The psyllid species carrying the bacteria can be considered as possible vectors of the bacterium (Teresani et al. 2014b). Electrical Penetration Graphs showed that B. trigonica was able to feed in the phloem of carrot, celery and potato but not in the phloem of tomato plants. Experimental transmission showed that B. trigonica transmitted ‘Ca. L. solanacearum’ from carrot to carrot, celery, potato and tomato. More efficient transmission occurred with ten individuals, and the transmission rates were 100% in celery, 80% in carrot and 10% in potato and tomato. The experimental transmission to potatoes threatens this crop (Teresani et al., 2014c). These combined results have built a scientific foundation of the biological and epidemiological aspects of ‘Ca. L. solanacearum’ contributing to new scientific information that is key in cultivation of celery and carrot to establish bacteria control strategies. The use of bacteria-free carrot seed lots will definitely contribute to mitigate damage and reduce risks of transmission to solanaceous crops. / Ribeiro Teresani, G. (2014). Candidatus Liberibacter solanacearum: detection, characterization, new hosts and epidemiology in Spain [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48459 / TESIS

Staphylococcal pathogenicity island SaPI1 is excised from genomic DNA and extrachromosomal copies are amplified during the vegetative growth of staphylococcal phage 80α. The amplified genetic element is subsequently encapsidated and transduced at very high frequency. Previous studies have demonstrated that the transducing particles have virions with tails that appear identical to those of helper phage 80α but have smaller capsids, commensurate with the smaller genome of the SaPI (Lindsay et.al., 1998). The morphology of the transducing particles, coupled with the observation that the genomic sequence of SaPIl (GenBank U93688) does not reveal any obvious phage structural proteins, has led to the hypothesis that SaPIl is encapsidated in a virion comprised of 80α structural proteins. Analysis of SaPIl transducing particles supports this hypothesis. Further investigation of 80α genes involved in SaPI1 mobilization was accomplished by selection of phage mutants resistant to SaPI1 interference. Two classes of SaPI1 jnterference resistant (sir) mutants were obtained, and point mutations were identified in two adjacent genes. In order to confirm the roles of these genes, an in-frame deletion of each candidate gene was constructed in an 80α prophage. All mutant phage and deletion constructs were evaluated for phage replication, SaPI1 replication, SaPIl transduction and SaPI1 interference. One gene (ORF21) was required for 80α growth and replication, but was not required for SaPIl growth or replication. The second gene (ORF22) was not essential for phage replication, but was required for SaPIl replication and high frequency transduction. The product of this gene was subsequently shown to be required for SaPIl excision.

O objetivo deste estudo foi avaliar a técnica de espectrometria de massas por ionização/dessorção a laser assistida por matriz tempo-de-vôo (MALDI-TOF) para a identificação direta em amostras de leite (sem cultivo microbiológico) de bactérias causadoras de mastite. Para tanto foram realizados dois experimentos (1 e 2). No experimento 1, o objetivo foi determinar a sensibilidade diagnóstica da técnica MALDI-TOF MS para a identificação direta em amostras de leite de Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae e Escherichia coli. Foram realizadas contaminações experimentais de S. aureus, S. uberis, S. agalactiae, S. dysgalactiae e E. coli em amostras de leite, para a obtenção de contagens de 103 a 109 ufc/mL. As amostras de leite contaminadas foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Espectros de massas foram coletados na faixa de massas de 2.000-20.000 m/z e foram analisados pelo programa MALDI Biotyper 3.0 (Bruker Daltonics) com as configurações padrão para obtenção da identificação bacteriana. A identificação direta de patógenos causadores de mastite a partir de amostras de leite foi possível em contagem ≥106 ufc/mL para S. aureus, ≥107 ufc/mL para E. coli, e ≥108 ufc/mL para S. agalactiae, S. dysgalactiae e S. uberis. No experimento 2, o objetivo foi avaliar o efeito da pré-incubação de amostras de leite de quartos mamários com mastite subclínica sobre a eficácia da identificação sem cultivo de patógenos causadores de mastite por MALDI-TOF MS. Foram selecionados 2 rebanhos leiteiros para coletas de leite de quartos mamários de todas as vacas em lactação. As amostras de leite foram submetidas às análises de: a) cultura microbiológica; b) pré-incubação seguida de identificação por espectrometria de massas diretamente do leite; c) contagem bacteriana total (CBT). Para a realização da CBT, as amostras de leite foram submetidas à citometria de fluxo; e para a identificação direta de patógenos causadores de mastite a partir do leite, as amostras foram submetidas ao desnate por centrifugação (10.000 Xg por 10 minutos), e pré-incubação a 37ºC por 12 horas. Posteriormente, as amostras foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Do total de 810 amostras de leite analisadas por cultura microbiológica (método referência), 347 apresentaram crescimento bacteriano, sendo 305 identificadas como agentes de interesse na identificação direta pelo método MALDI-TOF MS: Staphylococcus coagulase negativa (n=191), S. aureus (n=31), S. agalactiae (n=42), S. uberis (n=37), S. dysgalactiae (n= 4). Sendo assim, 305 amostras foram analisadas pelo método de idenfificação direta MALDI-TOF MS, a qual apresentou baixa sensibilidade quando comparado com a cultura microbiológica (método referência): Staphylococcus coagulase negativa (14,08%), S. agalactiae (15,25%), S. uberis (1,69%) S. aureus (6,12%) e S. dysgalactiae (0%). A pré-incubação de amostras de leite não aumentou a sensibilidade de identificação direta de microrganismos causadores de mastite pelo método MALDI-TOF MS. / The purpose of the present study was to evaluate the technique of mass spectrometry by desorption / ionization assisted laser array time-of-flight (MALDI-TOF MS) for the direct identification in milk samples (no microbiological culture) of mastitis causing bacteria. Therefore, we carried out two experiments (1 and 2). In experiment 1, we determined the diagnostic sensitivity of MALDI-TOF MS technique for the direct identification in milk samples of Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli. Experimental contamination of S. aureus, S. uberis, S. agalactiae, S. dysgalactiae and E. coli in samples of milk to a concentration of 103-109 cfu/mL were performed. The contaminated milk samples were processed using kit Maldi Sepsityper® (Bruker Daltonics) and subjected to bacterial lysis protocol for analysis by MALDI-TOF MS. Mass spectra were collected in the mass range of 2000-20000 m/z and were analyzed by MALDI Biotyper 3.0 software (Bruker Daltonics) with default settings to obtain bacterial identification. Direct identification of mastitis causing pathogens from milk samples was possible at ≥106 cfu/mL for S. aureus, ≥107 cfu/mL for E. coli and ≥108 cfu/mL for S. agalactiae, S. dysgalactiae and S. uberis. In experiment 2, the objective was to evaluate the effect of pre-incubation of milk samples from mammary quarters with subclinical mastitis on the effectiveness of identification without cultivation, of mastitis-causing pathogens by MALDI-TOF MS. We selected mammary quarter milk samples from all lactating cows on two dairy herds. The milk samples were subjected to analyzes of: a) microbiological culture; b) pre-incubation followed by identification by mass spectrometry directly from milk; c) total bacterial count (TBC). Flow cytometry was used to determine TBC and; to directly identify the mastitis-causing pathogens from milk, fat was separated by centrifugation (10,000 Xg for 10 minutes) and; samples were pre-incubated at 37°C for 12 hours. Subsequently, the skim milk samples were submitted to kit Maldi Sepsityper® (Bruker Daltonics) and to the bacterial lysis protocol for analysis by MALDI-TOF MS. A total of 810 milk samples were analyzed by microbiological culture (reference method), of which 347 showed bacterial growth. Considering all culture positive samples 305 were identified as agents of interest in the direct identification by MALDI-TOF MS method: coagulase negative staphylococci (n = 191), S. aureus (n = 31), S. agalactiae (n = 42), S. uberis (n = 37) and S. dysgalactiae (n = 4). Therefore, 305 samples were directly identified by MALDI-TOF MS, which presented low sensitivity when compared to microbiological culture (Reference method): coagulase-negative staphylococci (14.08%), S. agalactiae (15.25 %), S. uberis (1.69%), S. aureus (6.12%) and S. dysgalactiae (0%). Pre-incubation of milk samples did not increase the sensitivity of the MALDI-TOF MS method directly identify mastitis-causing microorganisms.

O presente trabalho teve como objetivo a caracterização da xilanase presente no extrato bruto de Bacillus pumilus, com a finalidade de usá-la na extração de xilana do bagaço de cana-de-açúcar. Foram testados dois meios de cultura em pHs 8,5 e 9,5 com diferentes substratos. As maiores atividades de xilanase foram produzidas em xilana comercial de oat spelts (228 U/mL) e em farelo de trigo (220 U/mL), no pH 8,5. Independentemente das condições de cultivo empregada, não foi detectada atividades de &szlig;-xilosidase, &szlig;-glicosidase e arabinofuranosidase e a produção de endoglucanase foi baixa (< 0,7 U/mL). O perfil de proteínas em eletroforese foi amplo, e a banda com 23 kDa correspondeu a uma xilanase. A xilanase apresentou pH ótimo na faixa de 7-8, temperatura ótima entre 45-50 ºC e se apresentou mais estável a 40 ºC (pH8,0). A hidrólise de xilanas comerciais produziu xilotriose, xilotetraose, xilopentaose e xilo-oligossacarídeos (XOS) com massas maiores, que não foram identificados por cromatografia em camada fina (TLC). A influência de vários fatores, tais como a carga de xilanase, tipo de substrato, temperatura e a adição de vários compostos foi avaliada no rendimento de extração de xilanas desde o bagaço de cana-de-açúcar prétratado. O extrato bruto de B. pumilus, rico em xilanases, foi aplicado em um bagaço pré-tratado com sulfito/álcali, e em um bagaço deslignificado por clorito em meio ácido, evidenciando que a menor quantidade de lignina residual como principal fator para aumentar a extração de xilana, aumentando a extração de 4,2 para 42,5%. A lavagem extensiva do material pré-tratado com sulfito alcalino também auxiliou no aumento do rendimento de xilana, de 18,5 para 25,1 %. Foram testadas outras alternativas para aumentar o rendimento de hidrólise da xilana do bagaço pré-tratado, sem a necessidade da lavagem do material. A reutilização do licor sulfito alcalino, a adição de tween 80, polietileno glicol, albumina não favoreceram a extração de xilanas, enquanto que a adição de MgSO4 teve um pequeno efeito positivo na extração da xilana (de 18% para 20,9 %). A extração alcalina a frio, realizada após a extração enzimática, influenciou positivamente o rendimento de extração da xilana do bagaço pré-tratado com 10% Na2SO3/5%NaOH. A extração de xilana obtido pela aplicação de xilanase (20 U/g) seguida da extração com 20% de NaOH produziu o mesmo resultado que utilizando apenas NaOH (70%), de 24,1 e 24,9 %, respectivamente. A extração enzimática da xilana permitiu a obtenção de um resíduo com características favoráveis para a completa sacarificação, com Cellic CTec2 na carga de 10 FPU/g de material. / The present work aimed to characterize the xylanase present in the crude extract of Bacillus pumilus, with the purpose of using it in the xylan extraction of the sugarcane bagasse. Two culture media were tested at pHs 8.5 and 9.5 with different substrates. The highest xylanase activities were produced in commercial xylan oat spelled (228 U / mL) and wheat bran (220 U / mL) at pH 8.5. Regardless of the culture conditions employed, &szlig;xylosidase, &szlig;-glycosidase and arabinofuranosidase activities were not detected and endoglucanase production was low (<0.7 U/mL). The protein profile on electrophoresis was extensive, and the 23 kDa band corresponded to a xylanase. The xylanase presented optimum pH in the range of 7-8, optimal temperature between 45-50ºC and was more stable at 40 ºC (pH8.0). Hydrolysis of commercial xylanes produced xylotriose, xylotetraose, xylopentaose and xylooligosaccharides (XOS) with larger masses, which were not identified by thin layer chromatography (TLC). The influence of various factors, such as xylanase loading, substrate type, temperature and the addition of various compounds was evaluated in the xylan extraction yield from the pretreated sugarcane bagasse. The crude extract of B. pumilus, rich in xylanases, was applied to a bagasse pretreated with sulfite/alkali, and to a bagasse delignified by chlorite in acid medium, showing that the lower amount of residual lignin as the main factor to increase the extracting xylan, increasing the extraction from 4.2 to 42.5%. Extensive washing of the alkaline sulfite pretreated material also assisted in increasing xylan yield, from 18.5 to 25.1%. Further alternatives were tested to increase the hydrolysis yield of pretreated bagasse xylan without the need for material washing. The reuse of the alkali sulfite liquor, the addition of tween 80, polyethylene glycol, albumin did not favor xylan extraction, while addition of MgSO4 had a small positive effect on xylan extraction (from 18% to 20.9%). The cold alkaline extraction, after enzymatic extraction, positively influenced the xylan extraction yield of the pretreated bagasse with 10% Na2SO3/5% NaOH. Extraction of xylan obtained by applying xylanase (20 U / g) followed by extraction with 20% NaOH produced the same result as using only NaOH (70%), 24.1 and 24.9%, respectively. The enzymatic extraction of xylan allowed to obtain a residue with favorable characteristics for the complete saccharification, with Cellic CTec2 in the load of 10 FPU / g of material.

O presente trabalho teve como objetivo a caracterização da xilanase presente no extrato bruto de Bacillus pumilus, com a finalidade de usá-la na extração de xilana do bagaço de cana-de-açúcar. Foram testados dois meios de cultura em pHs 8,5 e 9,5 com diferentes substratos. As maiores atividades de xilanase foram produzidas em xilana comercial de oat spelts (228 U/mL) e em farelo de trigo (220 U/mL), no pH 8,5. Independentemente das condições de cultivo empregada, não foi detectada atividades de &szlig;-xilosidase, &szlig;-glicosidase e arabinofuranosidase e a produção de endoglucanase foi baixa (< 0,7 U/mL). O perfil de proteínas em eletroforese foi amplo, e a banda com 23 kDa correspondeu a uma xilanase. A xilanase apresentou pH ótimo na faixa de 7-8, temperatura ótima entre 45-50 ºC e se apresentou mais estável a 40 ºC (pH8,0). A hidrólise de xilanas comerciais produziu xilotriose, xilotetraose, xilopentaose e xilo-oligossacarídeos (XOS) com massas maiores, que não foram identificados por cromatografia em camada fina (TLC). A influência de vários fatores, tais como a carga de xilanase, tipo de substrato, temperatura e a adição de vários compostos foi avaliada no rendimento de extração de xilanas desde o bagaço de cana-de-açúcar prétratado. O extrato bruto de B. pumilus, rico em xilanases, foi aplicado em um bagaço pré-tratado com sulfito/álcali, e em um bagaço deslignificado por clorito em meio ácido, evidenciando que a menor quantidade de lignina residual como principal fator para aumentar a extração de xilana, aumentando a extração de 4,2 para 42,5%. A lavagem extensiva do material pré-tratado com sulfito alcalino também auxiliou no aumento do rendimento de xilana, de 18,5 para 25,1 %. Foram testadas outras alternativas para aumentar o rendimento de hidrólise da xilana do bagaço pré-tratado, sem a necessidade da lavagem do material. A reutilização do licor sulfito alcalino, a adição de tween 80, polietileno glicol, albumina não favoreceram a extração de xilanas, enquanto que a adição de MgSO4 teve um pequeno efeito positivo na extração da xilana (de 18% para 20,9 %). A extração alcalina a frio, realizada após a extração enzimática, influenciou positivamente o rendimento de extração da xilana do bagaço pré-tratado com 10% Na2SO3/5%NaOH. A extração de xilana obtido pela aplicação de xilanase (20 U/g) seguida da extração com 20% de NaOH produziu o mesmo resultado que utilizando apenas NaOH (70%), de 24,1 e 24,9 %, respectivamente. A extração enzimática da xilana permitiu a obtenção de um resíduo com características favoráveis para a completa sacarificação, com Cellic CTec2 na carga de 10 FPU/g de material. / The present work aimed to characterize the xylanase present in the crude extract of Bacillus pumilus, with the purpose of using it in the xylan extraction of the sugarcane bagasse. Two culture media were tested at pHs 8.5 and 9.5 with different substrates. The highest xylanase activities were produced in commercial xylan oat spelled (228 U / mL) and wheat bran (220 U / mL) at pH 8.5. Regardless of the culture conditions employed, &szlig;xylosidase, &szlig;-glycosidase and arabinofuranosidase activities were not detected and endoglucanase production was low (<0.7 U/mL). The protein profile on electrophoresis was extensive, and the 23 kDa band corresponded to a xylanase. The xylanase presented optimum pH in the range of 7-8, optimal temperature between 45-50ºC and was more stable at 40 ºC (pH8.0). Hydrolysis of commercial xylanes produced xylotriose, xylotetraose, xylopentaose and xylooligosaccharides (XOS) with larger masses, which were not identified by thin layer chromatography (TLC). The influence of various factors, such as xylanase loading, substrate type, temperature and the addition of various compounds was evaluated in the xylan extraction yield from the pretreated sugarcane bagasse. The crude extract of B. pumilus, rich in xylanases, was applied to a bagasse pretreated with sulfite/alkali, and to a bagasse delignified by chlorite in acid medium, showing that the lower amount of residual lignin as the main factor to increase the extracting xylan, increasing the extraction from 4.2 to 42.5%. Extensive washing of the alkaline sulfite pretreated material also assisted in increasing xylan yield, from 18.5 to 25.1%. Further alternatives were tested to increase the hydrolysis yield of pretreated bagasse xylan without the need for material washing. The reuse of the alkali sulfite liquor, the addition of tween 80, polyethylene glycol, albumin did not favor xylan extraction, while addition of MgSO4 had a small positive effect on xylan extraction (from 18% to 20.9%). The cold alkaline extraction, after enzymatic extraction, positively influenced the xylan extraction yield of the pretreated bagasse with 10% Na2SO3/5% NaOH. Extraction of xylan obtained by applying xylanase (20 U / g) followed by extraction with 20% NaOH produced the same result as using only NaOH (70%), 24.1 and 24.9%, respectively. The enzymatic extraction of xylan allowed to obtain a residue with favorable characteristics for the complete saccharification, with Cellic CTec2 in the load of 10 FPU / g of material.

Xylella fastidiosa é uma bactéria gram-negativa que coloniza os vasos de xilema das plantas, podendo ser patogênica para algumas culturas de importância econômica. Esta bactéria é transmitida por cigarrinhas (Hemiptera: Auchenorrhyncha) da superfamília Cercopoidea (Aphrophoridae, Cercopidae e Clastopteridae) e família Cicadellidae (subfamília Cicadellinae). Na Itália este fitopatógeno esta causando uma nova doença em plantas de oliveira, denominada síndrome do declínio rápido da oliveira. Recentemente também foi detectada a ocorrência desta bactéria associada a sintomas de requeima foliar na Argentina e no Brasil. Sendo assim, surgiu a necessidade de realizar levantamentos de espécies de cigarrinhas que ocorrem nas regiões onde estão localizados os pomares de oliveira na Serra da Mantiqueira, nos estados de São Paulo e Minas Gerais, na faixa de 800 a 1800 m de altitude. O objetivo deste trabalho foi realizar a análise faunística das espécies de cigarrinhas, como também mensurar a similaridade da comunidade de espécies encontradas entre os pomares, e indicar possíveis vetores de Xylella fastidiosa para a cultura da oliveira. Foram selecionadas sete pomares de oliveira localizados na Serra da Mantiqueira, seguindo um gradiente altitudinal, sendo estes: Wenceslau Braz (MG-1780 m), São Bento do Sapucaí (Entre Vilas) (SP-1602 m), São Bento do Sapucaí (São José) (SP-1512 m), Maria da Fé (MG), com três pomares (área do Lago-1329 m, área da Suíça-1318 m, área da Atemoia-1310 m) e Cabreúva (SP-883 m). Para a captura de cigarrinhas, foram instalados em cada pomar nove cartões adesivos amarelos, em duas alturas, 0,8 m e 1,6 m do nível do solo, totalizando 18 cartões por pomar, que foram trocados a cada 15 dias no período de junho de 2015 a maio de 2017. Coletaram-se 40.324 indivíduos de 270 espécies incluídos em Auchenorrhyncha, distribuídos em 12 famílias, com maior frequência de indivíduos do grupo Cicadellidae (68%), e Cicadellinae (43% de todos os cicadelídeos coletados). A análise faunística indicou 44 espécies predominantes, sendo 20 pertencentes a grupos de potenciais vetores. Dentre elas, Clastoptera sp. 1 (Clastopteridae), Macugonalia cavifrons e Scopogonalia sp. 1 (Cicadellinae) foram predominantes em todos os pomares, exceto no pomar de Cabreúva, sendo que a primeira coloniza a oliveira. Os maiores índices de diversidade, riqueza e equitabiliade foram obtidos para os pomares de Wenceslau Braz, São Bento do Sapucaí (São José) e São Bento do Sapucaí (Entre Vilas) (0,87), respectivamente. A maior similaridade de Jaccard foi encontrada entre os pomares de Maria da Fé (55%). A maior similaridade de Morisita foi encontrada entre os pomares de São Bento do Sapucaí (São José) e Maria da Fé (área da Suíça) (91%) e a menor entre os pomares de Cabreúva e São Bento do Sapucaí (Entre Vilas) (6%). Para algumas espécies predominantes de Cicadellinae e Cercopoidea, houve diferença na eficiência de captura por armadilhas adesivas posicionadas em alturas distintas na copa das oliveiras. / Xylella fastidiosa is a gram-negative bacterium that colonizes the xylem vessels of plants and may be pathogenic to some crops. This bacterium is transmitted by leafhoppers (Hemiptera: Auchenorrhyncha) from the superfamily Cercopoidea (Aphrophoridae, Cercopidae and Clastopteridae) and family Cicadellidae (subfamily Cicadellinae). In Italy this phytopathogen is causing a new disease in olive trees, named \'olive quick decline syndrome\'. Recently, the occurrence of this bacterium associated to a \'leaf scorch\' was also detected in Argentina and Brazil. Thus, it is necessary to carry out surveys of Auchenorrhyncha species in olive orchards in the region of the Mantiqueira Mountain Range, where the olives are grown in the states of São Paulo and Minas Gerais, at 800 to 1800 m altitude range. The objectives of this work were to do faunistic analysis of Auchenorrhyncha species, to measure species similarity of the community of Auchenorrhyncha among orchards, and to indicate possible vectors of Xylella fastidiosa in olives. Seven olive orchards located in the Mantiqueira Mountain Range were selected, following an altitudinal gradient: Wenceslau Braz (MG-1780 m), São Bento do Sapuccaí (EntreVilas) (SP-1602 m), São Bento do Sapucaí (São José) (SP-1512 m), Maria da Fé (MG), with three orchards (area of Lago-1329 m, area of Suíça-1318 m, area of Atemoia-1310 m) e Cabreúva (SP-883 m). Nine yellow stick traps were installed in each orchard at two heights, 0.8 m and 1.6 m above soil level, totaling 18 cards per orchard, which were replaced fortnightly from June/2015 to May/2017. A total of 40,324 individuals from 270 species included in Auchenorrhyncha were collected, distributed in 12 families, with the highest frequency of individuals in the group Cicadellidae (68%) and group Cicadellinae (43% of all Cicadellidae). The faunistic analyses classified 44 species as predominant, 20 belonging to groups of possible vectors of X. fastidiosa. Among them, the species Clastoptera sp. 1 (family Clastopteridae), Macugonalia cavifrons and Scopogonalia sp. 1 (subfamily Cicadellinae) are predominant in all orchards, except in the Cabreúva orchard. Nymphs of Clastoptera sp. 1 were observed developing on young olive branches. Higher diversity, richness and equitability indices were observed in Wenceslau Braz, São Bento do Sapucaí (São José) and São Bento do Sapucaí (Entre Vilas) orchards, respectively. Jaccard\'s greatest similarity was found between Maria da Fé orchards (55%). The greatest similarity of Morisita was found between the orchards of São Bento do Sapucaí (São José) and Maria da Fé (91%) and the lowest one between orchards of Cabreúva and São Bento do Sapucaí (Entre Vilas) (6%). For some predominant species of Cicadellinae and Cercopoidea, capture efficiency was influenced by trap height on the olive tree canopy.