Bacterial degradation is an important way to detoxify environmental pollutants haloacids, and the key enzyme involved is dehalogenase. In contrast to the well characterized dehalogenases, haloacids transporters that mediate uptake of haloacids are poorly understood. The deh4p gene in a haloacids-degrading bacterium Burkholderia species MBA4 is the first reported haloacids transporter gene. It is located downstream of the dehalogenase gene deh4a and the two forms a haloacids operon. The role of Deh4p as a haloacids transporter was confirmed by heterologous expression. It was later found that a mutant of MBA4 without functional Deh4p was still able to grow in monochloroacetic acid (MCA), and Deh4p seems to be not the only haloacids transporter in MBA4. This study aimed at gaining a deeper understanding of the haloacids transport process in MBA4, and establishing the role of a newly identified gene dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. Disruption of deh4p in MBA4 caused a 32% decrease in MCA uptake rate, confirming the role of Deh4p as a haloacids transporter, but not the only one. A gene showing homology to deh4p in MBA4 was identified and named dehp2. The role of Dehp2 as a second haloacids transporter in MBA4 was confirmed by both gene disruption and heterologous expression. Like deh4p, the expression of dehp2 is also MCA-inducible. A double mutant with both deh4p and dehp2 disrupted only retained 36% MCA uptake rate, further confirming the roles of Deh4p and Dehp2 as haloacids transporters. Dehp2 and Deh4p were also shown to be the two major haloacids transporters. Both Dehp2 and Deh4p are specific toward acetate and its halogenated derivatives, but Dehp2 has a broader spectrum of substrates than Deh4p. Deh4p was found to be a better MCA transporter than Dehp2, and also has a higher affinity for MCA. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pH on MCA uptake supported the symport of proton(s) and the inclusion of both Dehp2 and Deh4p in the metabolites:H+ symporter family. Orthologs of dehp2 are widely found in the Burkholderia genus, and phylogenetic analysis showed that they were conserved in the genus. The roles of dehp2 orthologs in haloacids transport in three non-pathogenic Burkholderia species were studied. The three species were engineered to gain the ability to utilize MCA as the sole carbon source, and were shown to have MCA-inducible MCA uptake activities. Expressions of the dehp2 orthologs in them are MCA-inducible, strongly suggesting their relationship with MCA uptake. Fusion analysis with lacZ as a reporter gene confirmed the presence of MCA-inducible promoter activity in the upstream non-coding region of dehp2, and the results of electrophoretic mobility shift assay (EMSA) suggested a positive regulation of dehp2. This study established Dehp2 as a second haloacids transporter in MBA4, and also confirmed dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. The presence of at least two haloacids transporters in MBA4 demonstrated the complexity of this process, and future work should figure out the transport mechanisms. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-119) Also available in print.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Tobacco cyst nematodes (TCN), Globodera tabacum solanacearum, are one of the most serious pests for Virginia flue-cured tobacco (Nicotiana tabacum L.) farmers. These nematodes continue to spread to new farms every year and are now reported in 11 Virginia counties, seven North Carolina counties, and one farm in Maryland. The objectives of this study were to determine the ability of different populations of TCN to reproduce upon both a susceptible (K326) and resistant (NC567) cultivar, to compare TCN reproduction in non-infested soils with currently TCN-infested soils, and to examine reproduction and pathogenicity of TCN on other solanaceous crops. Tobacco cyst nematode reproduction on the TCN-resistant cultivar NC567 was significantly reduced when compared to the TCN-susceptible cultivar K326. Although significant differences among the populations were detected on both cultivars, the differences were not consistent across experiments. Results indicate similar pathogenicity among TCN populations on resistant and susceptible flue-cured tobacco cultivars. Plant breeders may use one population of the tobacco cyst nematode to screen tobacco germplasms for TCN resistance. Significant differences were noted in TCN reproduction among the various soils used in this study. However, the differences were inconsistent, making correlations between TCN reproduction and soil edaphic characteristics unreliable. Sterilizing the soils did not increase TCN reproduction. Reproduction in non-infested soils indicates that TCN will continue to spread throughout tobacco producing regions. Although traditionally an exclusive pest of tobacco, TCN's spread may threaten production of other solanaceous crops. Eggplant (Solanum melongena L.), pepper (Capsicum spp. L.), and tomato (Lycopersicon spp. L.) were confirmed to be hosts of TCN under field conditions. Eggplant was highly susceptible to TCN reproduction and yield suppression, exhibiting a 64% yield reduction in plots not treated with a nematicide compared to fumigated plots. Tomato allowed high rates of TCN reproduction but did not suffer yield loss. Tobacco cyst nematode reproduction was suppressed on pepper compared to the other two crops. No crop loss was noted in nontreated pepper plots compared to plots treated with a nematicide. Results would indicate that tobacco rotations including tomato or eggplant would be unacceptable due to high TCN reproduction rates. The tobacco cyst nematode does not seem to jeopardize the production of tomato and pepper in the southeastern United States, but does threaten profitability of eggplant production in this area. / Master of Science

Made available in DSpace on 2017-07-10T17:37:07Z (GMT). No. of bitstreams: 1 Luchele Furlan Sirtoli.pdf: 523026 bytes, checksum: d13d122671fc8cbbc6f1dbbdc76023c4 (MD5) Previous issue date: 2007-02-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Had been established 15 treatments from the combination of the Magali hybrid chili, grafeted in three rootstocks (AF 3001, AF 8251 and AF 8253, with their respective ungrafted plants) and inoculated with biovars I and III of Ralstonia Solanacearum. Each experimental area had four plants, being used all for evaluations. The used method of graft was of simple crack. Inoculum was constituted of bacterial cells obtained in saline solution from cultures in the logarithmic phase of growth. After the period of evaluations, the plants had been collected for isolation of the pathogen. For the grafted plants were collected three regions: region of the graft and regions above and below of the graft. For ungrafted plants, it was collected the region in the middle of the stem. There had no statistical differences for the height and diameter of fruits among treatments. Treatment AF 8251 - biovar III presented the greater number of commercial fruits for plant. The greater number of no commercial fruits was observed in the control treatment (ungrafted plants of Magali hybrid), to both biovars, and in hybrid AF 8353 - biovar III. Treatments AF 8251 - biovar I, AF 8251 - biovar III and the control treatment showed the best results for total mass of fruits per plant and total productivity. In the interaction rootstocks x regions of the plant, to biovar III, greater value of incidence are found in the region below of the graft in rootstocks AF 3001 / Foram estabelecidos 15 tratamentos, resultantes da combinação do híbrido de pimentão Magali em pé-franco e enxertado em três porta-enxertos (AF 3001, AF 8251 e AF 8253, com seus respectivos pés-francos), inoculados com os biovares I e III de Ralstonia Solanacearum. Cada parcela experimental contou com quatro plantas, sendo utilizadas todas para as avaliações. O método de enxertia utilizado foi o de fenda simples. O inóculo foi constituído de células bacterianas obtidas de culturas na fase logarítmica de crescimento, suspensas em solução salina. Após o período de avaliações, as plantas foram coletadas e levadas para laboratório para isolamento do patógeno. Para as plantas enxertadas foram coletadas três regiões: região da enxertia e regiões acima e abaixo desta. Para os pés-francos, coletou-se a região mediana da planta. Não houve diferenças estatísticas para altura e diâmetro médio de frutos entre os tratamentos. O tratamento AF 8251 - biovar III foi o que apresentou maior número de frutos comerciais por planta. Maior número de frutos não comercias foram observados na testemunha (pé-franco do híbrido Magali), tanto para o biovar I como para o biovar III, e no híbrido AF 8353 - biovar III. No que se refere à massa total de frutos por planta e a produtividade total, os tratamentos AF 8251 - biovar I e AF 8251 - biovar III, juntamente com a testemunha, apresentaram os melhores resultados. Na interação porta-enxerto x regiões da planta, para o biovar III, maior incidência foi encontrada na região abaixo da enxertia no porta-enxerto AF 3001

Ralstonia solanacearum est une bactérie phytopathogène à la gamme d'hôte exceptionnellement large et à la répartition mondiale. Cet organisme présente une biologie à facettes multiples et s'est adapté à quasiment tous les types de sols, à la vie planctonique, et à de nombreux hôtes et plantes réservoirs. Cette capacité d'adaptation est attestée par une très forte hétérogénéité des souches qui unifient ce complexe d'espèces, aussi bien au plan de la diversité génétique, phénotypique, que de la gamme d'hôte. Des approches phylogénétiques ont montré une structuration de la population mondiale en quatre phylotypes qui correspondent globalement à l'origine géographique des souches. Les travaux de thèse portent sur des souches du phylotype II qui ont valeur de modèle expérimental car épidémiologiquement inféodées à un hôte particulier : souches Moko pathogènes du bananier, souches ‘Brown rot’ adaptées à la pomme de terre et souches émergentes NPB, un variant du pouvoir pathogène. La question de recherche centrale porte sur la compréhension des mécanismes d'adaptation à l'hôte. Pour cela, une dizaine de génomes ont été séquencés dans une perspective (i) de revisiter la taxonomie de ce complexe d'espèce, (ii) d'en faire une analyse génomique comparative et (iii) d'analyser les paysages transcriptomiques produits lors de l'infection (in planta). L'ensemble des ces approches complémentaires permettent ainsi d'intégrer la complexité génétique et phénotypique de l'organisme de manière plus systémique. / Ralstonia solanacearum is a plant pathogenic bacterium globally distributed with a particularly broad host range. This organism is biologically diverse and is adapted to all types of soil, to planktonic lifestyle and to many plant hosts and natural reservoirs. This bacterium is a species complex and its genetic, phenotypic and host range diversity is a direct consequence of adaptation mechanisms. Phylogenetic analyses have divided this species complex into four distinct phylotypes correlating mostly with strains’ geographical origin. This thesis focuses on using phylotype II strains as an experimental model due to their adaptation to specific hosts: Moko strains pathogenic to banana, ‘Brown rot’ strains adapted to potatoes and emergent pathological variant NPB strains. Our main research topic is the understanding of host adaptation processes. In order to tackle this problematic we sequenced about ten genomes as a starting point of (i) a taxonomic revision of the species complex (ii) a comparative genomic analysis and (iii) an in planta transcriptomic analysis. Together, these complementary approaches allow a more systemic view of this organism’s genetic and phenotypic complexity.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-21T10:35:17Z No. of bitstreams: 1 Aldenir de Oliveira Alves.pdf: 1199354 bytes, checksum: 48b4ce9a3678bf554b211f23ec652d62 (MD5) / Made available in DSpace on 2017-02-21T10:35:17Z (GMT). No. of bitstreams: 1 Aldenir de Oliveira Alves.pdf: 1199354 bytes, checksum: 48b4ce9a3678bf554b211f23ec652d62 (MD5) Previous issue date: 2012-07-17 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The bacterial wilt caused by Ralstonia solanacearum (Rs) race 1, is an important bacterial disease in Pernambuco and other states of Brazil. This study aimed to evaluate the effectiveness of essential oils and silicon (Si) on alternative disease control, their effects on Rs, and verify the putative mechanisms involved in these processes. The oils of bergamot, copal, fennel, peppermint, sweet orange, lemon, cinnamon, lemon grass, eucalyptus citriodora, eucalyptus globulus, clary sage, and palmarosa were evaluated by biofumigation. Soil was previously infested with Rs (filotype I, race 1, biovar 3, biotype 8) at 5x108 CFU ml-1 and after seven days biofumigated during four days with the twelve oils (0.14%, v:v) under greenhouse conditions, and with bergamot, sweet orange and palmarosa in field. It was evaluated: the latency period (PL50), incidence, index of bacterial wilt (IBW) and area under the disease progress curve (AUDPC), plant development, Rs population in soil, soil characteristics, and Rs growth in vitro. The oils of palmarosa, bergamot and sweet orange elevated the PL50 and reduced IBW and AUDPC up to 15, 60 and 64.4%, respectively in greenhouse. In the field only palmarosa elevated the PL50 (38%), reduced the IMB (36%) and AUDPC (38%), and increased the number of fruits per plant. Only under greenhouse conditions soil biofumigated with oils of bergamot and sweet orange had higher sodium levels than those treated with palmarosa or control, which did not differ among them. In vitro, the growth of Rs was inhibited by the oil of palmarosa. To evaluate the effect of silicon (Si), sweet pepper plants cv. Enterprise were grown in compost containing 0.00, 0.25, 0.50, 1.50 and 3.00 g of SiO2 kg-1 of compost and transplanted into soil infested with isolate Rs CGM-8, being evaluated PL50, incidence, IMB and AUDPC; fresh and dry weight of shoot and root biomass, accumulation of Ca+2, Mg+2 and Si, total protein and enzymatic activity; chemical characteristics of substrate, and in vitro growth. The dose 3.00 g of SiO2 increased PL50 (34%), reduced IMB (63%) and AUDPC (47.4%), increasing also Ca+2 in shoots and reducing Mg+2 in shoots and roots. Supplementation with different Si doses resulted in maximum increments of fresh weight of shoot (121.8%), fresh weight of root (83.6%), and dry weight of shoot (84.9%); increased total protein, catalase, ascorbate peroxidase, and quitinase. Si was accumulated in the shoots and compost; pH, Na+ and K+ were elevated and P was reduced in the compost. The putative mechanisms of action of Si on disease control were direct action on the pathogen colonization, indirect action on plant growth, increase of Ca+2 absorption and signaling for producing plant defense enzymes. In conclusion, biofumigation with palmarosa oil and transplant production in compost containing calcium silicate are potential alternative strategies for the control of bacterial wilt of sweet pepper. / A murcha bacteriana do pimentão, causada por Ralstonia solanacearum (Rs) raça 1, é uma fitobacteriose importante em Pernambuco e outros estados do Brasil. Este trabalho teve como objetivos testar o efeito de óleos vegetais e do silício (Si) no controle alternativo desta doença, seus efeitos diretos sobre Rs e verificar os possíveis mecanismos envolvidos nestes processos. Os óleos essenciais de bergamota, canela, capim limão, copaíba, eucalipto citriodora, eucalipto globulus, funcho, hortelã, laranja doce, limão, sálvia esclareia e palmarosa foram avaliados por biofumigação. O solo foi previamente infestado com Rs CGM-8 (filotipo I, raça 1, biovar 3, biotipo 8) a 5x108 UFC ml-1. Após sete dias, o solo foi biofumigado durante quatro dias com os doze óleos essenciais (0,14%; v:v) em casa de vegetação, e com os óleos de bergamota, laranja doce e palmarosa em campo. Foram avaliados: período de latência (PL50), incidência, índice de murcha bacteriana (IMB) e área abaixo da curva de progresso da doença (AACPD); desenvolvimento da planta; população de Rs no solo; características do solo; e crescimento de Rs in vitro. Os óleos de palmarosa, bergamota e laranja doce elevaram o PL50 e reduziram IMB e AACPD em até 15, 60 e 64,4%, respectivamente, em casa de vegetação. Em campo, apenas o óleo de palmarosa aumentou o PL50 (38%) e reduziu IMB (36%) e AACPD (38%), elevando o número de frutos por planta. Apenas em casa de vegetação, solos biofumigados com os óleos de bergamota e laranja doce apresentaram maiores níveis de sódio que os tratados com palmarosa e testemunha, que não diferiram entre si. In vitro, o crescimento de Rs foi reduzido pelo óleo de palmarosa. O Si foi testado em pimentão cv. Enterprise cultivado em substrato contendo 0,00; 0,25; 0,50; 1,50 e 3,00 g de SiO2 kg-1 de substrato e transplantado para solo infestado com Rs CGM-8, avaliando-se: PL50, incidência, IMB, AACPD; biomassa; acúmulo de Ca+2, Mg+2 e Si; proteínas totais e atividade enzimática; características químicas do substrato e crescimento bacteriano in vitro. A dose 3,00 de Si aumentou o PL50 (34%) e reduziu IMB (63%) e AACPD (47,4%), aumentando ainda Ca+2 na parte área e reduzindo Mg+2 na parte aérea e raízes. A suplementação com diferentes doses de Si proporcionou incrementos máximos na biomassa fresca da parte área (121,8%), biomassa fresca da raiz (83,6%) e biomassa seca da parte aérea (84,9%); elevou proteínas totais, catalase, ascorbato peroxidase e quitinase; proporcionou acúmulo de Si na parte aérea e substrato; aumentou pH, Na+ e K+; e diminuiu P no substrato. Os prováveis mecanismos de ação do Si foram ação direta sobre a colonização do patógeno, ação indireta no desenvolvimento da planta, aumento na absorção de Ca+2 e sinalização para produção de enzimas de defesa da planta. Conclui-se que a biofumigação com o óleo essencial de palmarosa e a produção de mudas de pimentão em substrato contendo silicato de cálcio são práticas com potencial para o manejo alternativo da murcha bacteriana do pimentão.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-21T11:24:32Z No. of bitstreams: 1 Alessandra de Lima Garcial.pdf: 600161 bytes, checksum: ea17d29c4e5e21f8284154d25994db6d (MD5) / Made available in DSpace on 2017-02-21T11:24:32Z (GMT). No. of bitstreams: 1 Alessandra de Lima Garcial.pdf: 600161 bytes, checksum: ea17d29c4e5e21f8284154d25994db6d (MD5) Previous issue date: 2011-02-25 / Bacterial wilt caused by Ralstonia solanacearum is important worldwide mainly due to the great losses it causes in a large and diverse host range and its difficult control. In Brazil, bacterial wilt in solanaceous mainly occurs in Northeastern, North and Southeastern regions and may cause 100% losses. In the state of Pernambuco, bacterial wilt may be destructive to bell pepper in the producing counties of Mesorregiões Agreste and Mata. This work aimed: (i) to analyze 78 bacterial strains obtained from wilted bell pepper plants at producing counties of the state of Pernambuco, Brasil identified as R. solanacearum by multiplex PCR with reference to biochemical, virulence, molecular and genotypic diversity, and (ii) to evaluated the commercial disinfectant product Kilol- L® and its components, the ascorbic, citric and lactic acids for disease control. In the first paper the biochemical analysis characterized biovars and biotypes. The virulence studies included analysis of disease severity, area under disease progress curve and incubation period after inoculation of bell pepper, tomato, eggplant and tobacco plants by root wounding and inoculum drenching. The molecular characterization was performed by multiplex PCR, Rep-PCR and ISSR. The genotypic diversity was studied considering the number of observed genotypes and how they distributed by population differing in richness, evenness and diversity. There was predominance (97. 44%) of strains belonging to biovar 3, biotype 8 and phylotype I, but also were found biovar 1, biotypes 3 and 6, and phylotype II. The multivariate analysis performed with UPGMA and using the pool of the virulence data permitted the separation of R. solanacearum strains in 12 similarity groups, pointing out high variability, with strains of different areas in the same group. The Rep-PCR with REP and BOX markers and ISSR showed similarity among most of the R. solanacearum strains, although they did not separate strains belonging to biovars, biotypes, phylotypes, or from distinct areas. The genotypic diversity analysis evidenced moderate diversity in the total population but high variability among strains from the same county. In the second paper, initially it was evaluated in vitro the sensitivity of R. solanacearum to Kilol-L® (0.4, 0.8, 2.5, 5, 10 and 100%) and its components the ascorbic, citric and lactic acids (0.25, 0.5, 1, 2.5, 5 and 10%). After the phytotoxicity test in bell pepper cv. Atlantis, the concentrations Kilol-L® 0.8% and ascorbic, citric and lactic acids 1% were selected and tested for bacterial wilt severity reduction, by spraying of shoots or immersion of roots both using 21days-old plants. Ralstonia solanacearum strain CGH41 was inoculated by wounding roots and pouring inoculum. According to the analysis of the inhibition halos R. solanacearum showed high sensitivity to lactic acid 10% and medium sensitivity to lactic acid 5 and 2.5 %, ascorbic and citric acids 10% and Kilol-L® 100%. The immersion of roots in lactic acid 1% controlled bell pepper bacterial wilt by 100% however without difference from ascorbic and citric acids 1%. The method of root immersion differed from the shoot spray for most of the treatments. The unique significant effect of Kilol-L® was to elevate de incubation period when applied by shoot spraying. / A murcha bacteriana, causada por Ralstonia solanacearum tem importância mundial devido aos grandes prejuízos causados, à variada e extensa gama de hospedeiros e ao difícil controle. No Brasil, a murcha bacteriana das solanáceas ocorre principalmente nas Regiões Nordeste, Norte e Sudeste, podendo causar 100% de perdas. Em Pernambuco, a murcha bacteriana causa grandes prejuízos à cultura do pimentão nos municípios produtores das Mesorregiões do Agreste e Mata. Os objetivos deste trabalho foram: (i) analisar a diversidade bioquímica, da virulência, molecular e genotípica de 78 isolados obtidos de plantas de pimentão com murcha, em municípios produtores de Pernambuco, Brasil, identificados como R. solanacearum por PCR multiplex e (ii) avaliar o efeito do produto comercial Kilol-L® e seus componentes, ácidos ascórbico, cítrico e láctico, no controle da doença. No primeiro trabalho, na caracterização bioquímica foram determinados a biovar e o biotipo. Para o estudo da virulência, plantas de pimentão, tomate, berinjela e fumo foram inoculadas por ferimento de raízes e deposição do inóculo, analisando-se severidade, área abaixo da curva de progresso da doença e período de incubação. A caracterização molecular dos isolados foi realizada utilizando-se as técnicas de PCR-multiplex, Rep-PCR e ISSR. A diversidade genotípica foi estudada considerando o número de genótipos observados e como eles se distribuíam pela população, diferindo em riqueza, igualdade e diversidade. Nas populações estudadas foi observada a predominância (97,44%) de isolados da biovar 3, biotipo 8 e filotipo I, mas foram encontrados também a biovar 1, biotipos 3 e 6 e filotipo II. A análise multivariada por UPGMA, utilizando o conjunto dos dados de virulência,permitiu a separação dos isolados em 12 grupos de similaridade, indicando alta variabilidade, com isolados de diferentes áreas em um mesmo grupo. As análises de Rep-PCR, utilizando os marcadores REP e BOX, e ISSR, mostraram a existência de similaridade entre a maioria dos isolados de R. solanacearum. No entanto, esses marcadores não permitiram separar os isolados por biovares, biotipos, filotipos, nem por áreas diferentes. A análise da diversidade genotípica evidenciou diversidade moderada na população total, porém alta variabilidade dos isolados de um mesmo município. No segundo trabalho, inicialmente foi avaliada a sensibilidade in vitro de R. solanacearum ao Kilol-L® (0,4; 0,8; 2,5; 5; 10 e 100%) e seus componentes, os ácidos ascórbico, cítrico e láctico (0,25; 0,5; 1; 2,5; 5 e 10%). Após teste de fitotoxidez em plantas de pimentão ‘Atlantis’, as concentrações Kilol-L® 0,8% e ácidos ascórbico, cítrico e láctico a 1% foram selecionadas e testadas para redução da severidade da murcha bacteriana, aplicadas por pulverização da parte aérea ou pela imersão de raízes de plantas com 21 dias. Ralstonia solanacearum isolado CGH41 foi inoculado por ferimento de raízes e deposição do inóculo. A análise dos halos de inibição mostrou alta sensibilidade do patógeno ao ácido láctico 10% e média sensibilidade ao ácido láctico 5 e 2,5 %, ácidos ascórbico e cítrico 10% e Kilol-L® 100%. A imersão de raízes em ácido láctico 1% proporcionou 100% de controle da murcha bacteriana em pimentão, embora sem diferir estatisticamente dos ácidos ascórbico e cítrico 1%. O método de aplicação pela imersão de raízes diferiu da pulverização da parte aérea para a maioria dos tratamentos. O único efeito do Kilol-L® foi elevar o período de incubação, no método de pulverização.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.