Southern Blight (SB), caused by the fungus Athelia rolfsii, has increasingly impacted Mississippi soybean production with estimated economic losses in recent years rising from $181,616 in 2016 to $9,508,412 in 2021. Currently, there are no recommended fungicides and no known commercially available cultivar resistance. Eleven cultivars were evaluated for their response to the presence of Athelia rolfsii in the 2021 field trial. An in vitro fungicide assay was conducted to evaluate the efficacy of five fungicides to determine the respective EC50. These fungicides were then evaluated in combination with three cultivars, one mild, one moderate, and one severe in the 2022 field trial and in the growth chamber trial. A rapid and quantitative method was developed to evaluate the response of these commercially available soybean cultivars in combination with fungicides to SB. Determining effective management options has the potential to decrease disease losses for Mississippi Soybean growers.

No description available.

No description available.

Tobacco mosaic virus (TMV) is an extensively studied RNA virus that reduces quality and yield in commercially grown tobacco (Nicotiana tabacum L.). The virus is transmitted mechanically, although infections have been associated with contaminated seeds with the seed coat being the source of virus. Thus, TMV transmission is said to be seedborne (as opposed to true seed transmission where the embryo is infected). The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected K 326 flue-cured cultivar tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was developed to quantify viral titers in each component, while endpoint PCR confirmed RT- qPCR results and established a threshold viral cycle (Ct) value. Endpoint PCR results revealed viral accumulation in all three components of a tobacco seed. The highest concentration of TMV was in the SC, followed by ED and EM. A similar viral concentration gradient was observed in each individual tobacco seed from all three experimental plants. This is the first detection of TMV in tobacco embryos and suggests the virus can be seed transmitted. / Master of Science / Tobacco mosaic virus (TMV) is a widely studied plant virus that affects tobacco, tomato, pepper, and many other crops throughout the world. The virus is easily transmitted through contaminated tools or machinery, workers’ hands or clothing, or when an infected leaf comes into contact with an uninfected leaf. For years, TMV transmission was said to be seedborne, not seed transmitted, meaning that seedling infection comes from the infected external seed coat of the seed. Seed transmission of the virus has yet to be proven because of the difficulty to fully separate tobacco seed tissues. The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample, and synthesized into cDNA for analysis. A quantitative real-time polymerase chain reaction (RT-qPCR) assay was developed to quantify viral concentrations in each component. Endpoint PCR was used to confirm the quantitative results of RT-qPCR. Results revealed TMV accumulation in all three components of a tobacco seed, the highest concentration detected in the SC, followed by ED and EM; this pattern was observed from each plant. This is the first report of TMV being detected in embryos of tobacco seed which suggests that TMV can be seed transmitted.

The reduced capacity of antibiotics to treat infections is one of the greatest health concerns that society faces. There is substantial evidence that links this reduced capacity with the widespread use of antibiotics in livestock production. Livestock can act as reservoirs of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria, which can pass resistance on in the livestock's manure. It is important to understand the fate of antibiotic resistance genes and resistant bacteria in the environment after land-application of manure-based amendments. The goal of this field-scale study was to identify the effects of soil amendments (inorganic fertilizer, compost, or raw manure) and crop cover (lettuce or radish) on sediment transfer, fecal indicator bacteria (FIB), and release of ARGs in runoff over six storm events. Two FIB (Escherichia coli and enterococci) and two ARGs (sulI and ermB) were quantified in runoff from each of the constructed plots throughout the growing season. FIB and ARGs were recovered from all plots, including control plots indicating a background level within the soil. Additionally, only the effects of variability among individual storms had an impact on the concentration of FIB in runoff. Vegetative cover and storm variability affected sediment release. A trend of higher sul1 and ermB in runoff from compost and raw manure-amended plots for at least 2 months after planting crops was observed. Only one of these ARGs (ermB) is associated with the class of drugs given to the dairy cows used for the manure and compost, indicating inherent carriage of some ARGs independent of the type of antibiotic administered, and such genes can persist in the environment. These results suggest that there is a risk of ARGs being carried into areas downgradient from agricultural plots that have been amended with compost or manure. / MS

Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.

Les maladies fongiques du bois de la vigne que sont le syndrome de l'esca, le Black Dead Arm (BDA) et l'Eutypiose sont particulièrement dommageables à la profession vitivinicole, et sont actuellement en progression. Le temps d'incubation nécessaire à l'expression de ces maladies au champ complique l'évaluation de solutions préventives adaptées en condition contrôlée ainsi qu'en condition de terrain. Ces travaux de thèse ont eu pour objectifs la conception et la validation de tests PCR quantitatifs en temps réel (RtqPCR), permettant la détection et la quantification in planta de cinq champignons associés aux maladies de dépérissement de la vigne, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiose). Le développement de tests multiplexes a ensuite été entrepris et ces derniers ont été évalués pour la détection de quatre champignons (2 associés à esca et 2 au BDA) dans le bois de jeunes plants issus de pépinière viticole. Enfin, l'étude de l'interaction in planta de deux champignons associés au syndrome de l'esca de la vigne (P.chlamydospora et P.aleophilum) a été réalisée par RT-qPCR, et complétée par la caractérisation histologique de la réponse de la plante à la blessure dans le bois, en inoculation individuelle et en co-inoculation. / Grapevine trunk diseases, among which esca's syndrome, Black Dead Arm (BDA) and Eutypiosis, represent a real threat for grape and wine industry. Incubation time required before symptoms externalization in field complicates the evaluation of the efficacy of preventive solution in control and field conditions. These thesis's works focused on the design and the validation of Real Time quantitative PCR assays (RT-qPCR), in order to detect and quantify in vine plant five fungi associated with grapevine trunk disease, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiosis). The development of multiplex assays was undertaken and these last were evaluated in order to detect four fungi (esca and BDA) in wood sample from young plants in vine nursery. Finally, a study of the interaction between two fungi associated with esca's syndrome has been determined in planta through RT-qPCR, and completed by a histological analysis of plant response to injury of woody tissues.

L’orge (Hordeum vulgare L.) est l'une des céréales les plus importantes du monde et l’une des premières cultures domestiquées. Elle a été utilisée pendant des siècles pour l'alimentation humaine. Comme toutes les autres plantes, l'orge est dépendante de l'azote inorganique. L’efficacité de remobilisation de l'azote est donc très importante pour le remplissage des grains et pour la teneur en protéines du grain. L'objectif de ce travail est de donner une image du métabolisme des feuilles sénescence chez l'orge lorsque les plantes sont cultivées dans des conditions limitantes ou non en nitrates. Les analyses biochimiques, physiologiques et moléculaires de la sénescence des feuilles d'orge ont été réalisées. La gestion de l'azote pendant la sénescence des feuilles a été suivie par l'évolution des différents composés azotés au cours du vieillissement de la feuille. Une étude de profilage métabolique a été effectuée afin de déterminer les caractéristiques métaboliques de la sénescence des feuilles dans l'orge. En parallèle, les enzymes impliquées dans la remobilisation de l'azote ont été étudiées. Leurs activités et les niveaux de leurs transcripts ont été mesurés. Une attention particulière a été portée aux glutamine synthétases et asparagine synthétases et aux protéines de la machinerie de l'autophagie, processus connus pour jouer un rôle dans la remobilisation de l'azote pendant la sénescence des feuilles. A partir de toutes les données de séquences disponibles, ADNc, EST et séquences génomiques, cinq gènes codant pour les isoformes de glutamine synthétase cytosoliques (GS1), cinq gènes codant pour les isoformes d’asparagine synthétase (AS) isoformes et 19 gènes codant pour des protéines de la machinerie de l'autophagie ont été identifiés. Les expressions de tous les gènes identifiés ont été suivies au cours de la sénescence des feuilles et en fonction de l'alimentation en nitrates. La plupart de ces gènes sont sur-exprimés dans les feuilles sénescentes et de façon différentielle en fonction des conditions de nutrition. Toutes les données de séquences fournies par ce travail seront utiles à d'autres études translationelles et d'association génétique. / Barley (Hordeum vulgare L.) is one of the most important cereals in the world. It was one of the first domesticated crops and was used for centuries for human food. As all plants, barley has a fundamental dependence of inorganic nitrogen and nitrogen remobilization efficiency is very important for grain filling and grain protein content. The aim of this work was then to give a picture of the leaf-senescence metabolism in barley leaves when plants are grown under low or high nitrate conditions. Biochemical, physiological and molecular analyses of barley leaf senescence were performed. Nitrogen management during leaf senescence was monitored measuring changes in the different nitrogen pools during leaf ageing. In addition a large metabolite profiling study was performed in order to determine the metabolic hallmarks of leaf senescence in barley. In parallel enzymes involved in nitrogen remobilization were studied measuring their activity and the transcript levels of their coding genes. There was a special focus on glutamine synthetase and asparagine synthetase enzymes and for autophagy machinery that are known to play a role in nitrogen remobilisation during leaf senescence.From all the sequences data available, cDNA, EST and genomic sequences, we could identified five genes coding for cytosolic glutamine synthetase (GS1), five genes coding for asparagine synthetase (AS) and 19 genes coding for autophagy machinery proteins. Transcript levels of all the genes identified were monitored during leaf senescence and depending on nitrate nutrition. Most of these genes were over-expressed in senescing leaves and differentially expressed depending on nitrate conditions. In addition to the characterization of autophagy, GS1 and ASN genes, phylogenic and gene structures were analysed. All the sequences data provided by this work will be helpful to further translational and genetic association studies.

As áreas alagáveis naturais representam a mais importante fonte não-antropogênica de metano (CH4), com emissões estimadas entre 177 a 284 Tg ano-1, representando de 26 a 42% das emissões globais de CH4. A bacia do Rio Amazonas cobre uma grande porção dos trópicos úmidos, e a rede de drenagem deste rio excede a extensão de mais de um milhão de quilômetros quadrados. As grandes várzeas da bacia Amazônica são as maiores fontes naturais de CH4 desta região e estima-se que sua contribuição para as emissões totais de áreas alagadas no mundo seja na ordem de 5%. O CH4 produzido nas zonas anaeróbicas dos sedimentos por arquéias metanogênicas pode ser oxidado a CO2 pelos microrganismos metanotróficos. Com base na hipótese de que o fluxo de CH4 se altera sazonalmente em áreas alagáveis e que a microbiota presente está diretamente relacionada a esse processo, o presente estudo teve como objetivo geral avaliar a dinâmica dos genes funcionais envolvidos no ciclo do CH4 em épocas contrastantes, correlacionando com o fluxo do gás, variáveis ambientais e perfil taxonômico de Bacteria e Archaea em sedimentos de três áreas alagáveis e solo de floresta primária, da Amazônia Oriental (Belterra e Santarém-PA). Foram realizadas amostragem de gases, sedimentos e solo em duas épocas contrastantes (maio e outubro de 2016 - cheia e seca), para determinação da concentração de CH4 retido no sedimento durante a época cheia, cálculo do fluxo de CH4 durante a época seca, análises físico-químicas e extração de DNA dos sedimentos e solo para realização da qPCR dos genes funcionais mcrA e pmoA e dos genes marcadores filogenéticos 16S rRNA de Bacteria e Archaea, e sequenciamento do gene 16S rRNA de Bacteria e Archaea. A partir das amostragens de gases, foi possível observar que as áreas alagáveis possuem potencial de atuarem como fonte de CH4 durante a época cheia, e como fonte ou dreno de metano durante a época seca, confirmado pelas análises de qPCR, uma vez que a abundância do gene pmoA aumenta durante a época seca. Já no solo de floresta, o gene mcrA foi considerado como não detectado, portanto, a floresta pode ser considerada somente como potencial dreno de CH4. O estimador ACE e o índice Shannon mostraram que os sedimentos de áreas alagáveis possuem maior riqueza e diversidade de Bacteria e Archaea quando comparados ao solo de floresta. Todas as áreas apresentaram perfis taxonômicos do domínio Bacteria semelhantes, porém, a grande diferença entre as comunidades está relacionada ao domínio Archaea. A comunidade de arqueias no solo de floresta é majoritariamente composta por representantes do filo Thaumarchaeota. O solo de floresta apresentou baixa abundância dos filos potencialmente produtores de CH4, Bathyarchaeota e Euryarchaeota, e o contrário foi observado nas áreas alagáveis. Os dados gerados no presente estudo incentivam a continuidade de trabalhos relacionados ao ciclo do CH4 em áreas alagáveis da bacia Amazônica, incluindo investigações acerca do papel do filo Bathyarchaeota nessas áreas, principalmente em relação ao ciclo do CH4 / Natural wetlands represent the most important non-anthropogenic source of methane (CH4), with emissions estimated of 177-284 Tg year-1, accounting for 26-42% of global CH4 emissions. The Amazon basin covers a large portion of the humid tropics, and the drainage network of this river exceeds the extent of more than one million square kilometers. The wetlands of the Amazon basin are the largest natural sources of CH4 in this region and it is estimated that their contribution to the total emissions of wetlands in the world is around 5%. The CH4 produced in the anaerobic zones of the sediments by methanogenic archaea can be oxidized to CO2 by the methanotrophic microorganisms. Based on the hypothesis that methane flux changes seasonally in wetlands and its microbiota is directly related to this process, this research has the main objective to evaluate the dynamics of the functional genes involved in the CH4 cycle in contrasting seasons, correlating with the CH4 flux, environmental variables and taxonomic profile of Bacteria and Archaea in three wetlands and one primary forest of the Eastern Amazon (Belterra and Santarém-PA). The sampling of gas, sediments and soil was performed in May and October 2016 (wet and dry seasons) to determine the concentration of CH4 retained in the sediment in the wet season, measurement of CH4 flux in the dry season, physicochemical properties and molecular analysis (qPCR of the mcrA, pmoA functional genes and phylogenetic marker genes 16S rRNA of Bacteria and Archaea and sequencing of the 16S rRNA gene of Bacteria and Archaea). From gas samplings, it was possible to observe that wetlands have the potential to act as source of CH4 during the wet season, and as a source or drain of CH4 during the dry season, confirmed by qPCR analyzes, due the abundance increases of the pmoA gene during the dry season. In the forest soil, the mcrA gene was not detected, therefore, the forest could be considered only as CH4 drain potential. The ACE estimator and the Shannon index showed that the sediments of wetlands have higher richness and diversity of Bacteria and Archaea when compared to the forest soil. All areas presented similar taxonomic profiles of Bacteria, however, the main difference between the communities is related to the Archaea. The archaeal community in the forest soil is mostly composed of representatives of the phylum Thaumarchaeota. The forest soil presented low abundance of the phyla with potential CH4 producers, Bathyarchaeota and Euryarchaeota, however the opposite was observed in the wetlands. The data generated in the present study encourage the continuity of work related to the CH4 cycle in wetlands of the Amazon basin, including investigations about the role of the Bathyarchaeota phylum in these areas, especially in relation to the CH4 cycle

Objetivou-se com o presente estudo caracterizar as mudanças na população bacteriana ruminal, ocasionadas pelo aumento de concentrado na dieta, utilização de diferentes fontes de CNF e volumosos com diferentes digestibilidades da fibra. Para tanto, foram coletadas amostras de líquido ruminal, para posterior quantificação relativa de bactérias ruminais, oriundas de quatro projetos de pesquisa conduzidos no Laboratório de Pesquisa em Gado de Corte, pela FMVZ/USP - Pirassununga-SP. Em todos os experimentos, foram utilizados animais da raça Nelore, castrados e canulados no rúmen, em delineamento experimental de quadrado latino. Foi realizada uma quantificação relativa através da técnica de qPCR de três bactérias celulolíticas (Fibrobacter succinogenes, Ruminococcus albus e Ruminococcus flavefaciens), duas amilolíticas (Streptococcus bovis e Ruminobacter amylophilus), e uma consumidora de lactato (Megasphaera elsdenii), para determinação do efeito da dieta sobre a população de microrganismos ruminais. No Experimento 1, as dietas experimentais foram formuladas com dois níveis de concentrado: 60% ou 80%, sendo que o volumoso utilizado foi silagem de cana-de-açúcar (variedade IACSP 93-3046). Dentro de cada nível de inclusão de concentrado, foram utilizados três fontes de CNF: milho floculado a vapor (MFV), polpa cítrica peletizada (PCP), ou milho moído (MM). MFV e PCP foram incluídas na dieta em substituição parcial de 70% do MM. No Experimento 2, as dietas experimentais foram formuladas com 60% de concentrado, sendo que o volumoso utilizado foi a cana-de-açúcar fresca ou ensilada, com alta ou baixa digestibilidade da fibra (DFDN). No Experimento 3, as dietas experimentais foram formuladas com dois níveis de concentrado: 60% ou 80%, sendo que o volumoso utilizado foi a cana-de-açúcar fresca, com alta ou baixa DFDN. No Experimento 1, o aumento de concentrado resultou em queda da população de F. succinogenes (P<0,01) e S. bovis (P<0,01), e aumentou R. flavefaciens (P=0,05). A substituição parcial do MM por PCP resultou em aumento de S. bovis (P=0,01) e redução de R. flavefaciens (P<0,01). Já a substituição do MM por MFV reduziu R. albus (P<0,01). Houve uma interação Dieta*CNF apenas para a M. elsdenii (P=0,02), onde o MFV aumentou M. elsdenii apenas na dieta com 80% de concentrado. No Experimento 2, o fornecimento de cana fresca resultou em um aumento da população de S. bovis (P<0,01), e M. elsdenii (P=0,06). Houve interação entre DFDN e modo de conservação da cana sobre a população de F. succinogenes (P=0,01), onde a cana de alta DFDN aumentou a população de F. succinogenes apenas com o fornecimento de cana fresca. No Experimento 3, o aumento de concentrado resultou em queda de S. bovis (P<0,01), e aumento de R. amylophilus (P=0,07). Houve interação entre DFDN e nível de concentrado para a F. succinogenes (P=0,06) e R. albus (P<0,01), onde a cana de alta DFDN aumentou a população destes microrganismos apenas na dieta com 60% de concentrado. Com base nos resultados obtidos, conclui-se que o desempenho animal pode ser explicado pela modulação da população de microrganismos ruminais por meio da composição da dieta. / The aim of this study was to characterize the population change of cellulolytic and amylolytic rumen bacteria, caused by the increase of concentrate, and by the use of different sources of NFC in diets with sugarcane silage. Samples of rumen contents were collected for subsequent analysis of the relative quantification of rumen microorganisms, from four research projects conducted at the Research Laboratory in Beef Cattle at FMVZ / USP - Pirassununga-SP. In all experiments, Nellore beef cattle, castrated, and ruminal cannulated, were used in a Latin square design. Three cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens); two amylolytic (Streptococcus bovis and Ruminobacter amylophilus); and a lactate fermenting microorganism (Megasphaera elsdenii), were quantified by the technique of qPCR, to determine the effect of diet on the population of rumen microorganisms. Experiment 1, the experimental diets were formulated with two levels of concentrate: 60% or 80%, and the roughage used was sugarcane silage (IACSP 93-3046). Within each level of concentrate inclusion, three different sources of NFC were used: steam flaked corn (SFC), pelleted citrus pulp (PCP), or ground corn (GC). SFC and PCP were included in the diet in partial replacement of 70% of GC. Experiment 2, the experimental diets were formulated with 60% of concentrate level, and two sugarcane genotypes divergent for stalk NDFD, with high or low NDFD, either freshly cut or as silage. Experiment 3, the experimental diets were formulated with two levels of concentrate: 60% or 80%, and the roughage used was fresh sugarcane, with high or low NDFD. In the Experiment 1, increasing concentrate in the diet decreased the population of F. succinogenes (P<0.01) and S. bovis (P<0.01), and increased R. flavefaciens population (P=0.05). The partial replacement of GC by PCP increased S. bovis population (P=0.01) and decreased R. flavefaciens population (P<0.01). The replacement of GC by SFC decreased the population of R. albus (P<0.01). There was a significant Diet*NFC interaction only for M. elsdenii (P=0.02), where SFC increased the relative population only at the 80% concentrate diet. Experiment 2, Diets with fresh sugarcane increased the population of S. bovis (P <0.01), and M. elsdenii (P=0.06). There was a significant interaction between NDFD and conservation mode of sugarcane for F. succinogenes (P = 0.01), where sugarcane with high NDFD increased F. succinogenes population only when sugarcane was offered as freshly cut. In Experiment 3, the increase concentrate in the diet decreased S. bovis population (P<0.01), and increased R. amylophilus (P=0.07). There was a significant interaction between NDFD and concentrate level for F. succinogenes (P=0.06) and R. albus (P<0.01), where sugarcane with high NDFD increased the population of these microorganisms only at the 60% concentrate diet. The animal performance can be explained by modulation of the population of the rumen microorganisms through diet composition.