Anthracnose, incited by Colletotrichum cereale, is a destructive disease of physiologically stressed creeping bentgrass putting greens in Mississippi and Alabama. Anthracnose severity and frequency of occurrence have increased over the past 15 years, and fungicide resistance may have had a role in the increase. In vitro bioassays were performed to evaluate thiophanate methyl and azoxystrobin against C. cereale isolates exposed to the fungicides and baseline isolates that had not been exposed to either fungicide. All isolates collected from creeping bentgrass were uninhibited by both fungicides at discriminatory doses. Partial nucleotide sequences of the â-tubulin 2 (thiophanate methyl) or cytochrome b (azoxystrobin) gene was compared to confirm fungicide resistance. Thiophanate methyl resistance was conferred by either a point mutation from glutamic acid to alanine at position 198, or phenylalanine to tyrosine at position 200. Azoxystrobin resistance was conferred by an amino acid point mutation from glycine to alanine at position 143.

Antifungal drug resistance is a growing problem. Several mechanisms contribute to the development of resistance to antifungal agents. In South Africa, little is known about the antifungal susceptibility of local yeast isolates. The aim of this study was therefore to determine the antifungal susceptibility profile of local Candida species and Cryptococcus neoformans isolates and the molecular mechanisms of resistance to different antifungal agents in Pretoria. A total of 250 yeast isolates were collected from the diagnostic laboratory in the Department of Medical Microbiology at the University of Pretoria-National Health Laboratory Services. The isolates were subcultured on Sabouraud dextrose agar media for purity of yeast colonies. Identification to species level was performed using biochemical techniques. The antifungal susceptibility of 87 isolates was determined by the Etest for three azole antifungals (fluconazole, posaconazole and voriconazole), amphotericin B and caspofungin. Clinical breakpoint susceptibility was determined according to the CLSI clinical breakpoint reference methods. Polymerase chain reaction was performed on C. albicans isolates to amplify the ERG11 gene and the FKS1 gene. Sequencing was done for the amplification products and the sequence data were analysed by the CLC genome workbench software. Among the 250 isolates collected, Candida species accounted for 82.8% and C. neoformans accounted for 17.2% of the isolates. C. albicans was the most commonly isolated (76.8% of Candida species), of which 30% were resistant to caspofungin. Fluconazole resistance was detected in 56.7% of C. parapsilosis isolates, the highest fluconazole resistance among Candida species. Cross-resistance was found between fluconazole and voriconazole. Resistance to posaconazole was detected in 66.7% of C. glabrata isolates, whilst all other Candida species and C. neoformans isolates were fully susceptible. Furthermore, C. neoformans var. gattii isolates were less susceptible to azole antifungal agents than C. neoformans var. neoformans isolates. Molecular alterations are one of the mechanisms of resistance to azole and caspofungin antifungal agents. The amino acid substitutions D116E, K128T and V437I were detected in the ERG11 gene of two azole susceptible isolates. The new amino acid substitution E517Q was detected in the ERG11 gene of a resistant isolate. The S642L substitution was detected in the FKS1 gene of all the isolates that were caspofungin resistant and caspofungin susceptible. C. albicans was the most commonly isolated yeast species in Pretoria. Cross-resistance was detected between fluconazole and voriconazole. Therefore, these two agents are not a good alternative to use in the treatment of resistant isolates. With Candida species and C. neoformans isolates, there was less resistance to posaconazole than to fluconazole and voriconazole. The identification of the two varieties of C. neoformans is important in order to establish the differences in their antifungal susceptibility. Resistance to azole and caspofungin antifungal agents existed without the previously described molecular alterations in the ERG11 and FKS1 genes of resistant isolates. Further studies are required to explain the role of new amino acid substitutions, as well as the involvement of other mechanisms in resistance to antifungal drugs. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Medical Microbiology / unrestricted

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels.

Quinclorac controls crabgrass (Digitaria spp.) post-emergence in cool- and warm-season turfgrass. A rate response study revealed that two Mississippi smooth crabgrass (Digitaria ischaemum) species (MSU1 and MSU2) are resistant to quinclorac. Following that, field experiments were carried out to evaluate programmatic approaches to control one of these populations. Despite prior study on quinclorac-resistant weeds, to date, quinclorac-resistant smooth crabgrass and its mechanism of resistance have only been reported once in California. The mechanism of resistance of MSU1 and MSU2 relative to susceptible (SMT) was then investigated. The SMT biotype accumulated three times more cyanide than the resistant populations. Glutathione-S-transferase (GST) activity was evaluated as a possible contributor to non-target site resistance. The GST activity was elevated in the MSU1 and MSU2 populations. These findings suggest a non-target site–based mechanism of resistance involving the accumulation of cyanide. Further research is needed to investigate potential target-site mechanisms of resistance.

Recentemente, tem sido constatadas falhas no controle da planta daninha capim-branco (Chloris polydactyla) pelo herbicida glyphosate em áreas de culturas anuais e perenes no Brasil. Suspeita-se que estas falhas sejam decorrentes da seleção de populações resistentes desta planta daninha ao glyphosate. No entanto, até o momento, poucos relatos estão disponíveis na literatura caracterizando estas falhas. Esta pesquisa teve como objetivo a caracterização dos níveis de suscetibilidade de populações que apresentaram falha de controle, assim como estudar seus mecanismos de resistência. Também foi objetivo do trabalho analisar comparativamente o crescimento das populações suscetíveis e as supostamente resistentes ao glyphosate e avaliar a eficácia de herbicidas considerados alternativos ao glyphosate. Na primeira etapa, foram estudadas 87 populações do capim-branco vinda de diferentes regiões do Brasil, das quais duas sobreviveram ao experimento designado \"screening\". Esses dois biótipos, 59 e 69, juntamente com outras 15 populações suscetíveis, foram submetidos a experimentos de dose-resposta para a caracterização de suas suscetibilidades e determinação da dose eficiente de controle, da qual foi calculada em 705,41 g e.a. ha-1 de glyphosate. Em seguida, os biótipos supostamente resistentes foram comparados a um biótipo suscetível (34) para o cálculo do fator de resistência, que variou entre 3,92 e 7,95, confirmando a resistência dos biótipos. Não foi possível inferir claramente que existem diferenças de crescimento entre os biótipos resistente e suscetível que implicaria em custo adaptativo do biótipo resistente. No entanto, fica evidente que nas fases iniciais de crescimento, o biótipo 34S foi ou superior ou estatisticamente igual ao biótipo 69R. Não foi possível estabelecer uma relação entre diferenças de suscetibilidade entre os biótipos, portanto, a resistência do biótipo 69R ao glyphosate não alterou sua suscetibilidade aos herbicidas alternativos ao glyphosate aqui estudados. Na pós-emergência, clethodim e fenoxaprop-P-ethyl foram eficientes para controlar os biótipos 34S e 69R, enquanto que em pré-emergência, todos os herbicidas testados foram eficientes. O biótipo 69R absorveu menor quantidade de 14glyphosate em relação ao biótipo 34S em todos os tratamentos, do qual foi estatisticamente inferior àqueles contendo 14glyphosate + surfactante não-iônico (0,25%), 14glyphosate + sulfato de amônio (3kg ha-1) e 14glyphosate isolado. O tratamento que proporcionou maior absorção para ambos os biótipos foi o 14glyphosate + surfactante não-iônico. O biótipo 69R teve absorção máxima (Amax) 17% inferior ao biótipo 34S e maior retenção de 14glyphosate na folha tratada. O acúmulo de ácido chiquímico no biótipo 34S foi 2,56 vezes maior que no biótipo 69R. Mutações no gene que codificam a EPSPs dos biótipos 34S e 69R não foram observadas. Por fim, baseando-se nos dados aqui obtidos, é possível afirmar que a absorção reduzida, bem como a maior retenção do 14glyphosate nas folhas do biótipo 69R, possuem papel importante na resistência desse biótipo ao glyphosate. / It has been recently observed failures in controlling Tall windmill grass (Chloris polydactyla) by glyphosate in agricultural areas in Brazil. It is suspected that these failures are due to selection of resistant biotypes to glyphosate. However, to date, few reports are available in the literature characterizing these failures. Therefore, this study aimed to characterize the levels of susceptibility of differents populations of Chloris polydactyla, and to study the possible mechanisms of resistance to glyphosate. It was also an objective of this study to build a comparative growth analysis between populations, as well as evaluating the effectiveness of alternatives herbicides to glyphosate. In the first step, 87 populations from different regions of Brazil were studied, of which two survived the experiment called \"screening\". These two biotypes, 59 and 69, along with 15 other susceptible populations were subjected to a dose- response experiment for the characterization and determination of their susceptibility to glyphosate and a baseline herbicide sensitivity was obteined, which was calculated in 705.41 g ae ha-1 of glyphosate. Then the supposedly resistant biotypes (59R and 69R) were compared to a susceptible population (34S) for calculate the resistance factor, which ranged from 3.92 to 7.95, confirming the resistance of the biotypes. It was impossible to infer accurately that there are differences in growth between the resistant and susceptible biotypes of Tall windmill grass, which would imply in fitness penalty for the resistant biotype. However, it is evident that in the early growth stages, in most of the variables analyzed, the 34S was biotype or higher or statistically equal to biotype 69R. It was not possible to establish a statistical difference in susceptibility among biotypes, therefore the resistance of 69R biotype to glyphosate did not alter its susceptibility to alternative herbicides. At post-emergence, clethodin and fenoxaprop-P-ethyl were efficient to control biotypes 34S and 69R. On the other hand, at pre-emergence, all herbicides were efficient to control both biotypes. The biotype 69R absorbed smaller amount of 14glyphosate relative to 34S biotype in all treatments, which was statistically inferior to those containing 14glyphosate + non-ionic surfactant (0.25%), 14glyphosate + ammonium sulfate (3 kg ha-1) and 14glyphosate alone. The treatment providing greater absorption for both biotypes was 14glyphosate + non-ionic surfactant. Biotype 69R absorbed 17% less than the biotype 34S and had higher treated leaf retention. The shikimic acid accumulation assay in the biotype 34S was 2.56 times higher than in biotype 69R. Mutations in the gene encoding EPSPs were not observed. Finally, based on data obtained here, it is clear that the reduced absorption and treated leaf retention of 14glyphosate in biotype 69R, have an important role in the resistance of this biotype to glyphosate.

Recentemente, tem sido constatadas falhas no controle da planta daninha capim-branco (Chloris polydactyla) pelo herbicida glyphosate em áreas de culturas anuais e perenes no Brasil. Suspeita-se que estas falhas sejam decorrentes da seleção de populações resistentes desta planta daninha ao glyphosate. No entanto, até o momento, poucos relatos estão disponíveis na literatura caracterizando estas falhas. Esta pesquisa teve como objetivo a caracterização dos níveis de suscetibilidade de populações que apresentaram falha de controle, assim como estudar seus mecanismos de resistência. Também foi objetivo do trabalho analisar comparativamente o crescimento das populações suscetíveis e as supostamente resistentes ao glyphosate e avaliar a eficácia de herbicidas considerados alternativos ao glyphosate. Na primeira etapa, foram estudadas 87 populações do capim-branco vinda de diferentes regiões do Brasil, das quais duas sobreviveram ao experimento designado \"screening\". Esses dois biótipos, 59 e 69, juntamente com outras 15 populações suscetíveis, foram submetidos a experimentos de dose-resposta para a caracterização de suas suscetibilidades e determinação da dose eficiente de controle, da qual foi calculada em 705,41 g e.a. ha-1 de glyphosate. Em seguida, os biótipos supostamente resistentes foram comparados a um biótipo suscetível (34) para o cálculo do fator de resistência, que variou entre 3,92 e 7,95, confirmando a resistência dos biótipos. Não foi possível inferir claramente que existem diferenças de crescimento entre os biótipos resistente e suscetível que implicaria em custo adaptativo do biótipo resistente. No entanto, fica evidente que nas fases iniciais de crescimento, o biótipo 34S foi ou superior ou estatisticamente igual ao biótipo 69R. Não foi possível estabelecer uma relação entre diferenças de suscetibilidade entre os biótipos, portanto, a resistência do biótipo 69R ao glyphosate não alterou sua suscetibilidade aos herbicidas alternativos ao glyphosate aqui estudados. Na pós-emergência, clethodim e fenoxaprop-P-ethyl foram eficientes para controlar os biótipos 34S e 69R, enquanto que em pré-emergência, todos os herbicidas testados foram eficientes. O biótipo 69R absorveu menor quantidade de 14glyphosate em relação ao biótipo 34S em todos os tratamentos, do qual foi estatisticamente inferior àqueles contendo 14glyphosate + surfactante não-iônico (0,25%), 14glyphosate + sulfato de amônio (3kg ha-1) e 14glyphosate isolado. O tratamento que proporcionou maior absorção para ambos os biótipos foi o 14glyphosate + surfactante não-iônico. O biótipo 69R teve absorção máxima (Amax) 17% inferior ao biótipo 34S e maior retenção de 14glyphosate na folha tratada. O acúmulo de ácido chiquímico no biótipo 34S foi 2,56 vezes maior que no biótipo 69R. Mutações no gene que codificam a EPSPs dos biótipos 34S e 69R não foram observadas. Por fim, baseando-se nos dados aqui obtidos, é possível afirmar que a absorção reduzida, bem como a maior retenção do 14glyphosate nas folhas do biótipo 69R, possuem papel importante na resistência desse biótipo ao glyphosate. / It has been recently observed failures in controlling Tall windmill grass (Chloris polydactyla) by glyphosate in agricultural areas in Brazil. It is suspected that these failures are due to selection of resistant biotypes to glyphosate. However, to date, few reports are available in the literature characterizing these failures. Therefore, this study aimed to characterize the levels of susceptibility of differents populations of Chloris polydactyla, and to study the possible mechanisms of resistance to glyphosate. It was also an objective of this study to build a comparative growth analysis between populations, as well as evaluating the effectiveness of alternatives herbicides to glyphosate. In the first step, 87 populations from different regions of Brazil were studied, of which two survived the experiment called \"screening\". These two biotypes, 59 and 69, along with 15 other susceptible populations were subjected to a dose- response experiment for the characterization and determination of their susceptibility to glyphosate and a baseline herbicide sensitivity was obteined, which was calculated in 705.41 g ae ha-1 of glyphosate. Then the supposedly resistant biotypes (59R and 69R) were compared to a susceptible population (34S) for calculate the resistance factor, which ranged from 3.92 to 7.95, confirming the resistance of the biotypes. It was impossible to infer accurately that there are differences in growth between the resistant and susceptible biotypes of Tall windmill grass, which would imply in fitness penalty for the resistant biotype. However, it is evident that in the early growth stages, in most of the variables analyzed, the 34S was biotype or higher or statistically equal to biotype 69R. It was not possible to establish a statistical difference in susceptibility among biotypes, therefore the resistance of 69R biotype to glyphosate did not alter its susceptibility to alternative herbicides. At post-emergence, clethodin and fenoxaprop-P-ethyl were efficient to control biotypes 34S and 69R. On the other hand, at pre-emergence, all herbicides were efficient to control both biotypes. The biotype 69R absorbed smaller amount of 14glyphosate relative to 34S biotype in all treatments, which was statistically inferior to those containing 14glyphosate + non-ionic surfactant (0.25%), 14glyphosate + ammonium sulfate (3 kg ha-1) and 14glyphosate alone. The treatment providing greater absorption for both biotypes was 14glyphosate + non-ionic surfactant. Biotype 69R absorbed 17% less than the biotype 34S and had higher treated leaf retention. The shikimic acid accumulation assay in the biotype 34S was 2.56 times higher than in biotype 69R. Mutations in the gene encoding EPSPs were not observed. Finally, based on data obtained here, it is clear that the reduced absorption and treated leaf retention of 14glyphosate in biotype 69R, have an important role in the resistance of this biotype to glyphosate.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Conyza bonariensis, popularly known as fleabane, is a major weed of soybean crop in Southern Brazil, due to its evolution as resistant to glyphosate herbicide, high adaptability to production systems and high production of viable seeds. The aim of this work was to investigate the resistance to glyphosate in Conyza bonariensis, from the evaluation of the current level of resistance, alternative herbicides, aspects of growth and development of the resistant biotype when compared to the susceptible to the herbicide, as well as to determine the mechanism of resistance involved. The study was divided in three chapters, with their experiments carried out from May 2012 to July 2013. An experiment of dose-response curve was conducted to determine the level of resistance presented by fleabane. Concurrently, a study evaluated alternative herbicides to glyphosate. During the autumn-winter and spring-summer, there were conducted two experiments of fitness with biotypes susceptible (S) and resistant (R) to glyphosate, where were determined growth variables and yield and quality of seeds. Also, there were developed studies related and not realted to to the target site of the herbicide (EPSPS enzyme), aiming to elucidate the mechanism of glyphosate resistance involved to C. bonariensis specie. The level of resistance observed for fleabane biotype was high, with resistance factor >50. Alternative herbicides to glyphosate were effective in controlling fleabane, proving the non-occurrence of multiple resistance. The R biotype showed higher fitness to the S from variables analyzed, highlighting as superior in the plant height development and dry matter accumulation, besides to conclude the biological cycle in a shorter time than the S biotype. R plants also showed higher quantity and better quality of seeds produced. Thus, the R biotype tends to suppress the S one, even in the absence of the herbicide spray, disseminating the resistance and becaming more difficult the control of fleabane. From the studies related to the mechanism of glyphosate resistance, it is possible to infer the C. bonariensis R biotype shows changes involving EPSPS enzyme, target site of glyphosate, implying in its insensitivity to the herbicide enzyme. Therefore, glyphosate is not anymore a tool in the management of C. bonariensis, being necessary the use of alternative herbicides associated with integrated weed management measures. / Conyza bonariensis, popularmente conhecida como buva, é a principal planta daninha da cultura da soja no Sul do Brasil, em decorrência de sua evolução como resistente ao herbicida glyphosate, alta adaptabilidade aos sistemas produtivos e elevada produção de sementes viáveis. O objetivo desse trabalho foi investigar a resistência ao glyphosate em Conyza bonariensis, a partir da avaliação do nível de resistência atual, herbicidas alternativos, aspectos do crescimento e desenvolvimento de biótipo resistente quando comparado ao suscetível ao herbicida, bem como determinar o mecanismo de resistência envolvido. O estudo foi dividido em três artigos sendo seus experimentos desenvolvidos no período de maio de 2012 a julho de 2013. Um experimento de curva-de-dose-resposta foi conduzido para determinar o nível de resistência apresentado pela buva. Concomitantemente, um estudo avaliou herbicidas alternativos ao glyphosate. Durante o outono-inverno e primavera-verão, desenvolveram-se dois experimentos de valor adaptativo para os biótipos suscetível (S) e resistente (R) ao glyphosate, onde foram mensuradas variáveis de crescimento e, produção e qualidade de sementes. Também foram desenvolvidos estudos relacionados e não relacionados ao local de ação do herbicida (enzima EPSPS), buscando-se elucidar o mecanismo de resistência ao glyphosate envolvido para a espécie C. bonariensis. O nível de resistência observado para o biótipo de buva foi elevado, com fator de resistência superior a 50. Herbicidas alternativos ao glyphosate foram eficazes no controle do biótipo de buva, comprovando a não ocorrência de resistência múltipla. O biótipo R apresentou valor adaptativo superior ao S a partir das variáveis analisadas, destacando-se como superior no desenvolvimento em estatura e acúmulo de massa seca, além de concluir o ciclo biológico em menor período que o biótipo S. Plantas R também apresentaram maior quantidade e melhor qualidade de sementes produzidas. Deste modo, o biótipo R tende a suprimir o S, mesmo na ausência da aplicação do herbicida, dissiminando a resistência e dificultando ainda mais o controle da buva. A partir dos estudos relacionados aos mecanismos de resistência ao glyphosate, pode-se inferir que o biótipo R da C. bonariensis apresenta alterações envolvendo a enzima EPSPS, alvo do glyphosate, implicando em sua insensibilidade ao herbicida. Portanto, o glyphosate deixa de ser uma ferramenta no manejo da C. bonariensis, sendo necessária a utilização de herbicidas alternativos associado a medidas de controle integrado de plantas daninhas.

Mais de 20 milhões de hectares em todo o mundo são atualmente destinados a plantações de Eucalyptus, sendo que o Brasil possui a segunda maior área. No ano de 2007 a rede internacional EUCAGEN, liderada pelo Brasil, África do Sul e Estados Unidos, surgiu com o objetivo de colaboração para a pesquisa genômica do eucalipto. A árvore escolhida para o sequenciamento (Brasuz) foi fornecida pelo Brasil e em 2011 as primeiras sequências foram disponibilizadas. Em todas as fases de seu desenvolvimento, o eucalipto está sob o constante ataque de patógenos, destacando-se a ferrugem, causada pelo Basideomiceto Puccinia psidii Winter como a mais importante doença em regiões tropicais. A doença vem se espalhando rapidamente pelo mundo e recentemente foi relatada na Austrália, centro de origem do eucalipto. Com o objetivo de estudar o mecanismo molecular da interação plantapatógeno entre Eucalyptus grandis e Puccinia psidii, estabeleceu-se um patossistema modelo composto por um isolado monopustular do fungo e plantas resistente e susceptível provenientes de uma progênie de meios irmãos da planta Brasuz. O desenvolvimento do patógeno nos genótipos selecionados foi analisado por meio de microscopia de luz e de epifluorescência, e permitiu o monitoramento da dinâmica do desenvolvimento do fungo nos dois genótipos, com a identificação de todas as etapas de desenvolvimento do patógeno no genótipo susceptível bem como o estágio em que o genótipo resistente bloqueia o seu desenvolvimento. Com base nesses resultados determinou-se seis intervalos de interesse para a realização da análise da expressão gênica por meio da técnica de RNA-Seq. As análises revelaram grandes diferenças no perfil transcricional dos dois genótipos em resposta à presença do patógeno, permitindo a identificação de genes conhecidamente envolvidos em mecanismos de defesa de plantas como Receptores LRR-Quinase, fatores de transcrição WRKY, MYBS e GRAS, Proteínas R TIR-NBS-LRR Proteína Induzida por Injúria e proteínas envolvidas em processos de degradação proteica como F-Box. A comparação dos resultados provenientes das análises histológicas e moleculares permitiram a elaboração de um modelo para explicar os principais processos envolvidos no mecanismo de resistência de Eucalyptus grandis à Puccinia psidii. / More than 20 million hectares are destined to Eucalyptus plantations worldwide and Brazil has the second largest planted area. The International Eucalyptus Genome Network, EUCAGEN, was created in 2007 in order to perform genomic research on Eucalyptus. Brazil provided the biological material from the model tree (Brasuz) to have the complete genome sequenced and the first sequences were released to the scientific community in 2011. During Eucalyptus development, it is constantly exposed to pathogen attack with one of the most threatening diseases being eucalyptus rust caused by the neotropical rust fungus Puccinia psidii Winter which is rapidly spreading around the world and was recently described in Australia. In order to try and understand the molecular plant-microbe interaction between E. grandis and P. psidii we have to create a model system by isolating the pathogen from a single pustle and select resistant and susceptible plants from half-sib population generated using Brasuz as the pollen receptor. We performed light and epifluorescence microscopy analyses, identified all of the stages of the fungal development and recognized the moment which the resistant genotype blocks pathogen development. Based on these results we selected six time points to carry out transcriptomic analysis. Using RNA-Seq analysis we were able to verify large differences in transcriptional profile between resistant and susceptible plants and identify genes known involved in plant defense response such as LRR recptor Kinase, transcription factors (WRKY, MYBS and GRAS), TIR-NBS-LRR Proteins, Woundinduced protein and proteins involved in protein degradation (F-Box). Comparing microscopy and transcriptomic results allowed us to propose a model to explain the molecular mechanism of resistance of Eucalyptus grandis to Puccinia psidii.

Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...

Mais de 20 milhões de hectares em todo o mundo são atualmente destinados a plantações de Eucalyptus, sendo que o Brasil possui a segunda maior área. No ano de 2007 a rede internacional EUCAGEN, liderada pelo Brasil, África do Sul e Estados Unidos, surgiu com o objetivo de colaboração para a pesquisa genômica do eucalipto. A árvore escolhida para o sequenciamento (Brasuz) foi fornecida pelo Brasil e em 2011 as primeiras sequências foram disponibilizadas. Em todas as fases de seu desenvolvimento, o eucalipto está sob o constante ataque de patógenos, destacando-se a ferrugem, causada pelo Basideomiceto Puccinia psidii Winter como a mais importante doença em regiões tropicais. A doença vem se espalhando rapidamente pelo mundo e recentemente foi relatada na Austrália, centro de origem do eucalipto. Com o objetivo de estudar o mecanismo molecular da interação plantapatógeno entre Eucalyptus grandis e Puccinia psidii, estabeleceu-se um patossistema modelo composto por um isolado monopustular do fungo e plantas resistente e susceptível provenientes de uma progênie de meios irmãos da planta Brasuz. O desenvolvimento do patógeno nos genótipos selecionados foi analisado por meio de microscopia de luz e de epifluorescência, e permitiu o monitoramento da dinâmica do desenvolvimento do fungo nos dois genótipos, com a identificação de todas as etapas de desenvolvimento do patógeno no genótipo susceptível bem como o estágio em que o genótipo resistente bloqueia o seu desenvolvimento. Com base nesses resultados determinou-se seis intervalos de interesse para a realização da análise da expressão gênica por meio da técnica de RNA-Seq. As análises revelaram grandes diferenças no perfil transcricional dos dois genótipos em resposta à presença do patógeno, permitindo a identificação de genes conhecidamente envolvidos em mecanismos de defesa de plantas como Receptores LRR-Quinase, fatores de transcrição WRKY, MYBS e GRAS, Proteínas R TIR-NBS-LRR Proteína Induzida por Injúria e proteínas envolvidas em processos de degradação proteica como F-Box. A comparação dos resultados provenientes das análises histológicas e moleculares permitiram a elaboração de um modelo para explicar os principais processos envolvidos no mecanismo de resistência de Eucalyptus grandis à Puccinia psidii. / More than 20 million hectares are destined to Eucalyptus plantations worldwide and Brazil has the second largest planted area. The International Eucalyptus Genome Network, EUCAGEN, was created in 2007 in order to perform genomic research on Eucalyptus. Brazil provided the biological material from the model tree (Brasuz) to have the complete genome sequenced and the first sequences were released to the scientific community in 2011. During Eucalyptus development, it is constantly exposed to pathogen attack with one of the most threatening diseases being eucalyptus rust caused by the neotropical rust fungus Puccinia psidii Winter which is rapidly spreading around the world and was recently described in Australia. In order to try and understand the molecular plant-microbe interaction between E. grandis and P. psidii we have to create a model system by isolating the pathogen from a single pustle and select resistant and susceptible plants from half-sib population generated using Brasuz as the pollen receptor. We performed light and epifluorescence microscopy analyses, identified all of the stages of the fungal development and recognized the moment which the resistant genotype blocks pathogen development. Based on these results we selected six time points to carry out transcriptomic analysis. Using RNA-Seq analysis we were able to verify large differences in transcriptional profile between resistant and susceptible plants and identify genes known involved in plant defense response such as LRR recptor Kinase, transcription factors (WRKY, MYBS and GRAS), TIR-NBS-LRR Proteins, Woundinduced protein and proteins involved in protein degradation (F-Box). Comparing microscopy and transcriptomic results allowed us to propose a model to explain the molecular mechanism of resistance of Eucalyptus grandis to Puccinia psidii.