Microbiologia Agrícola
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/190
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Item Etiologia da podridão radicular e basal do caule da macaúba e potencial de fungos endofíticos radiculares no controle biológico da doença, com ênfase no estudo taxonômico de Dark septate endophytes (DSE)(Universidade Federal de Viçosa, 2025-02-25) Oliveira, Jaqueline Aparecida de; Pereira, Olinto Liparini; http://lattes.cnpq.br/7708215129316107Acrocomia aculeata é uma palmeira nativa da América do Sul e Central, com grande ocorrência no Cerrado. Essa planta pode produzir até 5-6 ton/ha de óleo a partir do fruto. Na última década, houve forte expansão do cultivo, especialmente no estado de Minas Gerais, consequência da demanda por matérias-primas que atendam a indústria de biocombustíveis. Um dos principais entraves para a produção da macaúba é a inexistência de estratégias de manejo bem definidas e de produtos específicos para a cultura, que garantam seu pleno desempenho no campo, mesmo sob as condições de estresse biótico e abiótico. O estabelecimento de planos de manejo mais eficientes passa pela elucidação da etiologia de doenças ainda desconhecidas que afetam a cultura. Paralelo a isso, estratégias sustentáveis para controle de agentes causais de doenças e promoção de crescimento da planta são ainda pouco exploradas nesta cultura. O objetivo da presente tese foi realizar o estudo de fungos endofíticos radiculares associados a macaúba e verificar seu potencial de aplicação no controle do principal agente causal da doença que afeta mudas da palmeira. Para isso, inicialmente foi elucidada a etiologia da podridão radicular e basal do caule que afeta mudas de macaúba em viveiros comerciais. Após, um estudo do potencial de uso dos fungos endofíticos radiculares associados a plantas sadias de macaúba no controle biológico de oomicetos foi conduzido. Em seguida, duas novas espécies e um novo gênero de fungos do tipo Dark septate endophytes (DSE) encontrados nas raízes sadias de macaúba foram descritos. E por fim, uma revisão sobre os fungos DSE encontrados em associação com a macaúba (e já relatados em outras culturas) é apresentada, destacando as características singulares destes fungos e os desafios e oportunidades de seu estudo. Esta tese faz parte da busca constante por compreender mais afundo todos os aspectos relacionados a cultura da macaúba. Além disso, traz insigths importantes sobre os potenciais usos de tecnologias biológicas a base de fungos que poderão ser aplicadas futuramente nos cultivos agrícolas. Palavras-chave: sustentabilidade Acrocomia aculeata; bioinsumos; novos taxa; oomicetos.Item Predição metagenômica, isolamento e caracterização de bactérias diazotróficas do solo de café arábica(Universidade Federal de Viçosa, 2025-02-25) Guimarães, Cleidiana Vieira; Silva, Marliane de Cassia Soares da; http://lattes.cnpq.br/1647689474581844Microrganismos diazotróficos ou promotores de crescimento de plantas (PGPR) contribuem para o fornecimento de nitrogênio assimilável (NH4?) as plantas. Investigações sobre esses grupos microbianos em culturas não leguminosas, como o café, oferecem insights promissores alinhados às demandas da agricultura de baixo impacto ambiental. Neste estudo, investigamos comunidades bacterianas associadas ao solo de duas fazendas produtoras de Coffea arabica. Realizou-se uma predição metagenômica de potenciais bactérias fixadoras de nitrogênio, com base nos genes estruturais nifHDK, vnfHDK e anfHDK, que codificam as isoformas Fe-Mo, Fe-V e Fe-Fe da nitrogenase. Paralelamente, foram realizados o isolamento, identificação molecular e testes in vitro com microrganismos cultiváveis dessas áreas. A análise metagenômica revelou a presença de táxons com genes de isoformas alternativas da nitrogenase no solo da Fazenda 2. Entre os gêneros isolados, destacaram-se Priestia, Dyella, Streptomyces e Luteibacter, com capacidade para fixação biológica de nitrogênio, produção de fitohormônios e solubilização de fosfato inorgânico. Os resultados indicam que a comunidade de bactérias diazotróficas do solo é dinâmica e que abordagens metagenômicas combinadas com o isolamento microbiano se complementam, permitindo não apenas a identificação de diversidade genética funcional latente, mas também a possibilidade de validação do potencial biotecnológico de diazótrofos de vida livre em cafeeiros. Palavras-chave: Fixação Biológica de Nitrogênio; Predição funcional; Microbiota do Solo.Item RNAi em Colletotrichum lindemuthianum: caracterização das proteínas-chave e desafios para a aplicação(Universidade Federal de Viçosa, 2025-03-25) Rosa, Rafael Oliveira; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/8163562962122113O agente etiológico da antracnose, uma das principais doenças do feijão-comum (Phaseolus vulgaris L.), é o fungo Colletotrichum lindemuthianum. Devido à alta variabilidade genética do patógeno e as limitações das estratégias convencionais de controle, como o uso de cultivares resistentes e fungicidas, torna-se necessário o desenvolvimento de novas abordagens eficientes e sustentáveis. Neste contexto, o RNA de interferência (RNAi) surge como uma alternativa promissora, permitindo o silenciamento de genes essenciais para a patogenicidade do fungo. Com isso, o presente estudo teve como objetivos: caracterizar os principais componentes da maquinaria de RNAi em C. lindemuthianum; avaliar a expressão dos genes-chave durante a interação com o feijão-comum; investigar a capacidade do fungo de captar dsRNAs exógenos; e silenciar genes-alvo quando aplicados por pulverização (SIGS). A análise do genoma de C. lindemuthianum permitiu identificar proteínas- chave da maquinaria de RNAi, incluindo duas Argonautas (AGO), duas Dicer-like (DCL) e três RNA polimerases dependentes de RNA (RdRPs). Dentre essas, ClAGO2, ClRdRP2 e ClRdRP3 foram descritas pela primeira vez na espécie. Todas as proteínas apresentaram domínios altamente conservados, e a análise filogenética indica que os genes que as codificam foram mantidos nos genomas, com maior proximidade entre espécies do mesmo complexo taxonômico do gênero Colletotrichum. A expressão dos genes AGO1, RdRP3 e DCL1 do fungo na interação com a planta variou ao longo do tempo e da linhagem avaliada. Foram obtidos 18 e 180 transformantes de C. lindemuthianum e C. truncatum, respectivamente, expressando GFP. A produção de dsRNAs apresentou baixo rendimento em dois métodos testados (in vivo e in vitro), resultando em uma média de 12,2 µg de dsRNA sintetizado pelo kit comercial. Os ensaios de captação de dsRNA por microcultivo indicaram que C. lindemuthianum é capaz de perceber dsRNAs exógenos, com aumento no número e tamanho de vacúolos, porém os resultados foram inconclusivos quanto a absorção. A pulverização dos dsRNAs direcionados a ClSGE1 (regulador transcricional putativo) e ClpacC (regulador transcricional em resposta ao pH) a 4 ng.µL-1 não foram capazes de controlar a antracnose no feijão-comum nas condições avaliadas. Dessa forma, foi possível caracterizar as proteínas envolvidas no mecanismo de RNAi e demonstrar que C. lindemuthianum é capaz de perceber dsRNAs exógenos, abrindo caminho para futuros testes de captação. Além disso, este estudo contribui para a compreensão dos desafios associados a padronização e o uso de dsRNA como estratégia de controle de fitopatógenos e inicia as investigações desse método para C. lindemuthianum. Palavras-chave: RNAi; Antracnose; Feijão-comum; SIGS.Item Development and evaluation of chimeric recombinant antigen, nano vaccine and probiotics for fish to Aeromonas hydrophila and Pseudomonas fluorescens control(Universidade Federal de Viçosa, 2025-02-26) Rodrigues, Jojo; Mendes, Tiago Antonio de Oliveira; http://lattes.cnpq.br/9161106617289234Aquaculture is an aquatic food production system whose main products are the cultivation of fish, bivalves and algae. It contributes approximately US$230 billion to the world economy annually. Growth estimates project trends until 2050 and, therefore, aquaculture is considered one of the most promising global markets. The high consumption of fish in the human diet has intensified in recent years due to the benefits it brings to human health and nutrition, since fish are a source of proteins, vitamins, minerals and “good” fats, which help prevent cardiovascular, bone, ophthalmological and degenerative diseases. However, fish production has been threatened by the establishment of pathogenic bacteria that cause a wide range of diseases and comorbidities in fish and are associated with high mortality rates and production losses in aquaculture production. The species Aeromonas hydrophila and Pseudomonas fluorescens are representatives of a group of Gram-negative bacteria that threaten fish health and have an alarming zoonotic potential. Vaccination of fish, as well as alternative methods such as the use of probiotics in supplementation, are considered the gold standard in the fight against these infectious agents. Therefore, this study aimed to develop and evaluate a chimeric, nanoencapsulated, probiotic antigen for fish against pathogenic strains of Aeromonas hydrophila and Pseudomonas fluorescens. The antigen was constructed using immunoinformatics tools using the sequence of the protein UDP-N-acetylmuramate-L-alanine ligase, which is important for the biogenesis of the precursor of the cell wall of bacteria, as a skeleton. A prediction of B and T cell epitopes was performed on the BepiPred 2.0 and NetCLT web servers, and the most immunogenic protein sequences of both bacteria were used to construct the chimeric antigen. The evaluation of the immunogenic property was evaluated in the animal model, zebrafish (Danio rerio), and the gene expression of genes related to the fish immune system was evaluated. Protection after immunization was evaluated by challenging the LD50 or lethal doses of A. hydrophila in vivo and survival was recorded over a period of seven days after challenge. The nano antigen was also nanoencapsulated by nanostructured lipid nanocarriers and the physicochemical characteristics of the formulations as well as the immunogenic and protective properties in vivo were evaluated. In addition, the antimicrobial and probiotic potential of two strains, Lactococcos lactis and Lactobacillus acidophilus, was performed and characterized in vivo. The results were promising and demonstrated an ease of production of the antigen on a large scale through expression in bacterial systems, as well as an immunogenic and protective potential culminating in the upregulation of genes of the fish immune system and in the high survival percentage of immunized fish that were challenged with the LD50 and lethal doses of A. hydrophila. The results were even more promising when the antigen was nanoencapsulated, demonstrating that after nanoencapsulation, the protection and survival of the fish were significant at low concentrations of the antigen and challenge of the fish with a highly lethal dose of A. hydrophila. However, the probiotic and antimicrobial evaluation of the strains of L. lactis and L. acidophilus was positive only in vitro, culminating in a direct antimicrobial activity against pathogenic strains of A. hydrophila and P. fluorescens, but the same was not observed in vivo, and the protection and survival of the fish that received the probiotic treatments were not significant in relation to the fish that were not treated. The data presented in this work demonstrate the power of using immunoinformatics and nanotechnology for the construction and production of products with antimicrobial and vaccine potential against pathogenic agents. Keywords: Aquaculture; Fish; Pathogenic bacteria; Aeromonas hydrophila; Vaccination; Immunoinformatics.Item Isolation and characterization of foodborne pathogen-infecting phages and genetic determinants of phage susceptibility in Staphylococcus aureus(Universidade Federal de Viçosa, 2025-05-21) Cunha, Paloma Cavalcante; Paula, Sergio Oliveira de; http://lattes.cnpq.br/1842375517176650The growing prevalence of antibiotic-resistant bacterial pathogens in clinical, veterinary, and food production settings demands alternative control strategies. Bacteriophages (phages), due to their host specificity and safety, have emerged as promising antimicrobial agents for both therapeutic and biocontrol applications. This thesis explores the isolation, genomic and functional characterization, and application of lytic phages targeting Salmonella enterica and Staphylococcus aureus, two major foodborne and zoonotic pathogens. The study also investigates bacterial genetic determinants that modulate susceptibility to phage infection. In Chapter 1, the polyvalent Tequintavirus phage UFVCit2 was isolated and shown to infect Citrobacter freundii, Shigella flexneri, and different S. enterica serovars. Genomic analysis confirmed its strictly lytic nature and genetic safety. UFVCit2 significantly reduced S. enterica counts on chicken meat and lettuce in food model experiments, highlighting its potential for biocontrol applications in complex matrices. Chapter 2 presents the characterization of phage CapO46, a newly identified Rosenblumvirus with broad activity against S. aureus strains from clinical and environmental origins. CapO46 exhibited rapid adsorption kinetics, a short latent period, and stable lytic activity in ultra-high-temperature (UHT) milk, achieving up to 7.2 log10 CFU/mL reduction after 12 hours. Genomic analyses confirmed the absence of lysogeny, virulence, or resistance genes, further supporting its suitability as a safe and effective biocontrol agent for dairy applications. In Chapter 3, a genome-wide CRISPR interference (CRISPRi-seq) screen was conducted to identify host factors in S. aureus that influence susceptibility to CapO46. The screen revealed that repression of mgrA, sarZ, and SAOUHSC_00695 conferred resistance to infection, while silencing rpiRc increased susceptibility. Functional validation via bacterial growth, phage replication, adhesion, and biofilm formation assays confirmed the involvement of these genes in regulating both phage sensitivity and surface-associated virulence traits. Altogether, this work demonstrates the potential of phages as targeted, safe, and sustainable alternatives to antibiotics, and underscores the importance of genetic context in modulating bacterial susceptibility. The integration of phage genomics, food model applications, and functional bacterial genetics provides a comprehensive framework for the rational development ofphage-based biocontrol and therapeutic strategies. Keywords: Salmonella enterica; Staphylococcus aureus; Biocontrol; CRISPR interference; Host factors; Phage resistance.Item Efeito do etanol, cloreto de sódio e peróxido de hidrogênio sobre o crescimento de linhagens de Spathaspora passalidarum isoladas do bioma amazônico(Universidade Federal de Viçosa, 2025-02-26) Andrade, Julia Martins de; Fietto, Luciano GomesO Brasil é o segundo maior produtor de etanol do mundo. O uso de combustíveis sustentáveis tem sido cada vez mais desejável visando menor impacto ambiental. Como forma de suprir a demanda energética sem utilizar hidrocarbonetos e derivados agrícolas, o Etanol de Segunda Geração (E2G) surge como uma possibilidade. No caso deste biocombustível, os microrganismos e as biomassas lignocelulósicas exercem um papel crucial. Por isso, este trabalho teve como objetivo caracterizar fisiologicamente 12 linhagens da levedura Spathaspora passalidarum isoladas do bioma brasileiro amazônico, visando futuras aplicações na indústria sucroalcooleira. Esta espécie possui potencial para a produção de E2G por ser fermentadora de xilose. Os experimentos avaliaram o perfil de crescimento das 12 linhagens em estresses provocados por etanol (5%, 6% e 7%), cloreto de sódio (0,5 M, 0,75 M e 1 M), peróxido de hidrogênio (7 mM e 10 mM) e diferentes concentrações de glicose (2%, 18%, 20%,25% e 30%). Os ensaios de estresse foram realizados em meio YPD e meio YPX utilizando o equipamento Growth Profiler 960 (EnzyScreen). Ademais, foram realizados dois experimentos de co fermentação glicose/xilose a fim de identificar fontes de carbono preferenciais e rendimento de produção de etanol (g/L). Os resultados apontaram que o perfil de crescimento das linhagens apresentou variações de acordo com o estresse e o tipo de fonte de carbono (glicose ou xilose). Em meio YPX (xilose), as linhagens apresentaram maior resiliência ao estresse por etanol. Em condições de estresse por NaCl, a levedura de referência obteve o maior crescimento em 1 M, com OD (600 nm) de aproximadamente 1,2. Na primeira cofermentação, observou-se consumo preferencial de glicose (esgotada em até 12 horas por todas as linhagens). A linhagem 6 demonstrou consumo eficiente de xilose e produziu 10,5 g/L de etanol em 24 horas. A segunda cofermentação foi realizada com a levedura NRRL Y-27907 e o a levedura isolada número 6, sendo o isolado mais bem sucedido na produção de etanol. Portanto, conclui-se que a linhagem 6 de S. passalidarum apresentou melhor desempenho em relação aos outros isolados nos ensaios realizados. Palavras-chave: Leveduras; Spathaspora passalidarum; Resposta ao estresse; Produção de etanol; Biocombustíveis; Etanol 2G.Item Bioprospection and adaptive mechanisms of halotolerant bacteria: applications in biotechnology and astrobiological studies(Universidade Federal de Viçosa, 2025-02-25) Vieira, Camila de Souza; Totola, Marcos Rogerio; http://lattes.cnpq.br/9880193425166992Halophilic and halotolerant bacteria can be applied in environments where conventional organisms cannot thrive, such as in the bioremediation of salinized soils or the treatment of saline effluents. These organisms are also frequently used in astrobiology studies due to their well-recognized ability to tolerate or adapt to extreme conditions. Brazil, with its diverse biomes and high biodiversity, harbors saline environments that remain largely unexplored in terms of microbial biodiversity. This study aimed to investigate the biotechnological potential and the feasibility of using halotolerant bacteria isolated from extreme Brazilian environments (arid and saline) as model organisms in astrobiological studies, as well as to expand the understanding of the adaptive capacity of halotolerant microorganisms to NaCl. The bacteria were collected from three areas in Camocim, Ceará, Brazil. Their identification was carried out through fatty acid methyl ester (FAME) profiling and the analysis of the DNA sequence encoding the 16S rRNA. The requirement for or tolerance to NaCl and sodium perchlorate (NaClO4) were evaluated by determining the specific growth rates (µ) in culture media containing different salt concentrations. The ability to use NaClO4 as a final electron acceptor was also assessed. The biotechnological potential of the isolates was evaluated by analyzing their production of biosurfactants, exopolysaccharides, and the enzymes lipase, protease, and amylase. To study the adaptive capacity of microorganisms to sodium chloride concentrations above the maximum originally tolerated by the wild-type strain, the technique of adaptive laboratory evolution (ALE) was applied. The study used one of the isolates collected in Camocim, selected based on its µ value at 100 g L-1 of NaCl, and a bacterium previously isolated from Trindade Island, Brazil, which had demonstrated high adaptability to NaCl. The evolved and parental/wild-type strains were compared using growth curves and specific growth rate values at different NaCl concentrations. Additionally, it was assessed whether the mutations generated by ALE altered the growth rate in the presence of NaClO4 as a stressor and whether they affected the fatty acid profile in response to NaCl concentration in the growth medium. Among the isolates, those belonging to the genus Bacillus were predominant (18/20). All isolates were able to grow at 100 g L-1 NaCl and at least 30 g L-1 NaClO4, in addition to being able to reduce NaClO4 in the absence of other electron acceptors. The isolates could produce biosurfactants, exopolysaccharides, and enzymes of industrial/biotechnological interest. ALE resulted in variants with a higher specific growth rate and greater tolerance to higher NaCl concentrations compared to the parental strains. Furthermore, an increase in µ at 70 g L-1 NaClO4 was observed, along with changes in the fatty acid profile under both saline and non-saline stress conditions. Keywords: extremophiles; astrobiology; salt adaptation; adaptive laboratory evolution.Item Promotion of microbial phosphate solubilization by clay minerals(Universidade Federal de Viçosa, 2024-08-23) Iglesias Barrera, Gustavo Andrés ; Costa, Maurício Dutra; http://lattes.cnpq.br/8820351730014631A number of physical and chemical interactions between clays and microorganisms have been reported with strong influence on microbial physiology and ecology. However, clay influences on phosphate-solubilizing microorganisms (PSM) remains unknown. PSM are capable of converting low solubility phosphates into soluble orthophosphate that can be taken up by plants. PSM are commonly present in the soil, particularly in the rhizosphere where they maintain associative symbiosis with the host plant. This study aimed at investigating the influence of soil clays (kaolinite, gibbsite, goethite, and hematite) on phosphate solubilization by the fungus Aspergillus niger. In the first chapter, a literature review is presented addressing the potential use of PSM in agriculture. The interaction between clays and microorganisms is reported and the strategies to enhance microbial phosphate solubilization are described. In the second chapter, an investigation on the influence of kaolinite, gibbsite, goethite, and hematite, on organic acid production and phosphate solubilization by the fungal isolate A. niger FS1. The experiments were conducted in modified NBRIP medium containing 3 g L! of Araxá rock phosphate (RP) supplemented with clays at 250, 500, 1000, 1500, 2000, 2500, and 3000 g L'!. The media were inoculated with 10º fungal conidia and incubated for 7 days at 28ºC. AII clays promoted increases in Araxá RP solubilization. The addition of gibbsite and kaolinite to the culture media led to the highest values of soluble P recorded (453.68 and 407.61 mg L', respectively). Gibbsite increased oxalic acid production by the fungus 8.36 times that of the control treatment. Gibbsite and kaolinite also promoted higher yields of citric acid, with 15.69 and 8.93 mmol L' ', respectively. In the third chapter, the hypothetical mechanisms of promotion of phosphate solubilization by clays were verified. Clays were added directly to the culture media as described above or placed in plastic capsules to prevent direct contact of the particles with the fungus. The effects of increasing concentrations of Fe-EDTA, AlCls, and silicic acid on Araxá RP solubilization and organic acid production were also evaluated. The encapsulation of clay particles did not decrease RP solubilization, indicating that the direct contact of the clays with the fungal mycelium is not required to induce RP solubilization. Significant increases in soluble P (3.89 times that of the control treatment) and oxalic acid were observed with increasing concentrations of AlCl;. Fe-EDTA and silicic acid increased phosphate solubilization by only 29 and 11%, respectively, with no significant increases in oxalic and citric acid production. In the fourth chapter, we investigated the effect of kaolinite, gibbsite, goethite, and hematite on the solubilization of Araxá, Argélia, Bayovar, Catalão, Marrocos, and Patos de Minas RPs and aluminum, calcium, and iron phosphate. For this, modified NBRIP medium was supplemented with 3 g L! of each P source above. The media were inoculated with 10º fungal conidia and incubated for 7 days at 28ºC. Gibbsite was the most efficient clay at promoting the solubilization of all RPs tested, reaching 100% cof solubilization for the majority. Kaolinite was particularly effective at the solubilization of iron phosphate, reaching 80.9% of solubilization. Only goethite was capable of enhancing aluminum phosphate solubilization (22./2%). In the fifth chapter, a brief review is presented discussing the question: Are there really microorganisms specialized in phosphate solubilization or is this process merely a side-effect of other microbial metabolic activities? All the findings presented here offer new possibilities for developing innovative strategies to enhance RP solubilization using soil clays. Our results shed light on the interactions between soil clays and fungal hyphae that may operate in the soil to increase P availability to plants. They also improve our understanding on P dynamics in the soil involving PSM and clay minerals. Keywords: Soil clays, phosphate solubilization, organic acids, kaolinite, gibbsite, goethite, hematite, phosphate solubilizing microorganisms.Item Actinobactérias como agentes de biocontrole e promoção de crescimento de soja(Universidade Federal de Viçosa, 2024-08-26) Fernandes, Fábulo Junior Nogueira; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/2541389172357294Item Penicillium sp. 658F5R-AM endofítico de Hevea brasiliensis: anotação do genoma, controle de fitopatógenos, promoção de crescimento vegetal e produção de metabólitos secundários(Universidade Federal de Viçosa, 2024-08-24) Silva, Mirele Lopes da; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/9323296572880336