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Brazil is the second-largest producer and the first exporter of beef, with herds mainly raised in extensive pastures, where Megathyrsusmaximus occupies over 30 Mha. About 70% of the pastures are under degradation, and using plant growth-promoting bacteria (PGPB) may contribute to reversing this scenario. We investigated the effects of PGPB on the growth of six cultivars of M. maximus—Tanzania-1, Massai, BRS Zuri, Mombaça, BRS Tamani, and BRS Quênia—under greenhouse conditions. Plants were inoculated, or not, with the elite strains of Azospirillum brasilense CNPSo 2083 + CNPSo 2084, Bacillus subtilis CNPSo 2657, Pseudomonas fluorescens CNPSo 2719, or Rhizobium tropici CNPSo 103. At 35 days after emergence, plants were evaluated for ten root growth traits, shoot dry weight, and the levels of macro and micronutrients accumulated in shoots. Several root traits were increased due to inoculation in all genotypes, impacting plant growth and nutrient uptake. Despite the differences in effectiveness, all genotypes benefited from PGPB to some degree, but Mombaça and BRS Zuri were more responsive. Scanning electron microscopy indicated that bacterial species differed in their capacity to colonize seeds and rootlets. The results show that inoculation with elite PGPB strains may represent an important strategy for the sustainability of M. maximus pastures.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

Aspergillus spp. are ubiquitous fungi that grow on stored grains. Some species produce toxins that can harm human and animal health, leading to hepato- and nephrotoxicity, immunosuppression and carcinogenicity. Major fungicides used to prevent fungal growth may be toxic to humans and their repeated use over time increases levels of resistance by microorganisms. Nanotechnology is an emerging field that allows use of antimicrobial compounds in a more efficient manner. In this study, was evaluated the antifungal activity of biogenic silver nanoparticles (AgNPs, synthesized by fungi) and simvastatin (SIM, a semi-synthetic drug), alone and in combination against three toxigenic species belonging to the genera Aspergillus section Flavi (Aspergillus flavus, Aspergillus nomius and Aspergillus. parasiticus) and two of section Circumdati (Aspergillus ochraceus and Aspergillus melleus). SIM exhibited a MIC50 of 78 μg/mL against species of Section Flavi and a MIC50 of 19.5 μg/mL against species of Section Circumdati. The MIC50 of AgNPs against Aspergillus flavus, Aspergillus nomius and Aspergillus parasiticus was 8 μg/mL, while the MIC50 was 4 μg/mL against Aspergillus melleus and Aspergillus ochraceus. Checkerboard assay showed that these compounds, used alone and in combination, have synergistic and additive effects against toxicogenic species of Aspergillus. Analysis by SEM gives an idea of the effect of SIM and AgNPs alone and in combination on spore germination and vegetative growth. Ultrastructural analysis revealed that spore germination was prevented, or aberrant hyphae were formed with multilateral branches upon treatment with SIM and AgNPs. These results reveal potential benefits of using combination of AgNPs and SIM to control fungal growth.

Candida tropicalis has emerged as one of the major Candida non-C. albicans species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis pathogenic mechanism has yet not been fully defined. The current study aimed to demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms were produced on small catheter fragments (SCF) and were mainly composed by blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces (HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of blastoconidia involved by extracellular matrix. Total biomass and metabolic activity from the remaining biofilm on SCF surface, after direct contact with human cells, exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular matrix components, after contact with human cells. Thus, we described for the first time an easy and simple in vitro model with catheter, which could be a powerful tool for future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.

The objective of this study was to evaluate the antibacterial effect of sophorolipid in combination with lactic acid against relevant bacteria isolated from the poultry industry. Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli were isolated from chicken meat and antibacterial tests with sophorolipid and lactic acid were performed. Checkerboard, time-kill, and scanning electron microscopy analyses were used to confirm the antibacterial action and the combined effects. Although no inhibitory effects were observed for E. coli and Salmonella, these compounds presented antibacterial activity against L. monocytogenes and S. aureus. Additionally, sophorolipid and lactic acid were not cytotoxic at the concentrations used in the tests. The combination of sophorolipid and lactic acid resulted in an additive interaction, reducing the concentration of the active compounds needed for effectiveness against S. aureus and L. monocytogenes, to 50% and 75%, respectively. These findings lead to the possibility of developing a new, sustainable, and natural antimicrobial solution that is considered noncytotoxic and has wide applicability in the poultry industry to reduce substantial losses in this sector.

Vulvovaginal candidiasis (VVC) is a common vaginitis that affects women, especially in childbearing age, caused byCandida albicansin almost 80% of cases. Considering the limited drug arsenal available and the increasing fungal resistance profile, the search for new therapeutic sources with low toxicity and easy administration should be supported. Propolis has been used as a traditional medicine for multiple diseases, considering its particular composition and pharmaceutical properties that permits its wide applicability; it has also emerged as a potential antifungal agent. Thus, this study performed anin vitroandin vivoinvestigation into the efficacy of a new mucoadhesive thermoresponsive platform for propolis delivery (MTS-PRPe) in a preclinical murine model of VVC treatment caused byC.albicans. The methodologies involved chemical analysis, an assessment of the rheological and mucoadhesive properties of propolis formulations,in vitroandin vivoantifungal evaluations, histological evaluations and electron microscopy of the vaginal mucosa. The results demonstrated the antifungal activity of propolis extract and MTS-PRP against the standard strain and a fluconazole-resistant clinical isolate ofC.albicans, in bothin vitroandin vivoassays. These results were similar and even better, depending on the propolis concentration, when compared to nystatin. Thus, the formulation containing propolis exhibited good performance againstC.albicans in a vulvovaginal candidiasis experimental model, representing a promising opportunity for the treatment of this infection.

Candida krusei is one of the most common agents of invasive candidiasis and candidemia worldwide, leading to high morbidity and mortality rates. This species has become a problem due to its intrinsic resistance and reduced susceptibility to azoles and polyenes. Moreover, the number of antifungal drugs available for candidiasis treatment is limited, demonstrating the urgent need for the discovery of novel alternative therapies. In this work, the in vivo and in vitro activities of a new oxadiazole (LMM11) were evaluated against C. krusei. The minimum inhibitory concentration ranged from 32 to 64 μg/mL with a significant reduction in the colony forming unit (CFU) count (~3 log10). LMM11 showed fungicidal effect, similar to amphotericin, reducing the viable cell number (>99.9%) in the time-kill curve. Yeast cells presented morphological alterations and inactive metabolism when treated with LMM11. This compound was also effective in decreasing C. krusei replication inside and outside macrophages. A synergistic effect between fluconazole and LMM11 was observed. In vivo treatment with the new oxadiazole led to a significant reduction in CFU (0.85 log10). Furthermore, histopathological analysis of the treated group exhibited a reduction in the inflammatory area. Taken together, these results indicate that LMM11 is a promising candidate for the development of a new antifungal agent for the treatment of infections caused by resistant Candida species such as C. krusei.

Poincianella pluviosa has already been described as capable of healing skin wounds. In an attempt to prolong contact of the drug with the wound, it was proposed in this study to evaluate wound healing using a crude extract (CE) of P. pluviosa incorporated in carboxymethylcellulose polymer films. The chromatographic profile of the semipurified fraction of P. pluviosa was evaluated by ultra-high performance liquid chromatography (UHPLC), confirming the compounds gallic acid, geraniin, and ellagic acid. The films were evaluated for their physical and mechanical properties, water vapor permeability, moisture absorption capacity, and FTIR spectroscopy. For in vivo experiments, wounds were made on the back of rats and treated daily for 4, 7, 10, or 14 days with film containing CE or control film. At the end of each period, skin permeation analysis and histological analysis were made using re-epithelialisation, cell proliferation, and collagen formation. Statistical significance was determined by GraphPad Prism using t test and Mann-Whitney test. Anti-staphylococcal activity was evaluated with standard strains of Staphylococcus aureus, methicillin-resistant, and coagulase negative. It was demonstrated that the presence of CE in the films increased the capacity to absorb water and decreased resistance and permeability. The CE of the film permeated the skin, reaching the dermis and was able to influence re-epithelisation, cell proliferation, and collagen formation. Satisfactory results were observed against S. aureus strains, particularly coagulase negative. Films with CE of P. pluviosa can be an alternative in the wound healing, protecting against opportunistic infections and giving comfort to the patient.

Microplastics (MPs) are critical emerging pollutants found in the environment worldwide; however, its toxicity in aquatic in amphibians, is poorly known. Thus, the aim of the present study is to assess the toxicological potential of polyethylene microplastics (PE MPs) in Physalaemus cuvieri tadpoles. According to the results, tadpoles’ exposure to MP PE at concentration 60 mg/L for 7 days led to mutagenic effects, which were evidenced by the increased number of abnormalities observed in nuclear erythrocytes. The small size of erythrocytes and their nuclei area, perimeter, width, length, and radius, as well as the lower nucleus/cytoplasm ratio observed in tadpoles exposed to PE MPs confirmed its cytotoxicity. External morphological changes observed in the animal models included reduced ratio between total length and mouth-cloaca distance, caudal length, ocular area, mouth area, among others. PE MPs increased the number of melanophores in the skin and pigmentation rate in the assessed areas. Finally, PE MPs were found in gills, gastrointestinal tract, liver, muscle tissues of the tail and in the blood, a fact that confirmed MP accumulation by tadpoles. Therefore, the present study pioneering evidenced how MPs can affect the health of amphibians.

The toxicity of zinc oxide nanoparticles (ZnO NPs) has been addressed in several studies; however, their effect on the mammalian group, even at environmentally relevant concentrations, remains poorly understood. The aims of the present study are to expose female Swiss mice to ZnO NP concentrations commonly faced by mammals who enter aquatic systems to perform different ecological functions and to assess the possible effects of such particles on their behavior. The test animals were placed in water added with ZnO NPs for 3 min, 2 times/day, for 21 days. Two experimental groups were set, NP1x, composed of animals subjected to ZnO NP concentration of 760 μg/L; and NP50x (control), which encompassed animals subjected to 38,000 μg/L. Based on field test results (OF), the contact with NPs did not induce locomotor deficits or anxiogenic and anxiolytic effect on the animal models. However, models exposed to NPs were not able to recognize the predatory threat posed by the presence of Pantherophis guttatus and Arapaima gigas; on the other hand, animals in the control group, who were not exposed to ZnO NPs, did not present antipredator behavioral response deficit. Furthermore, mice exposed to NPs were unable to distinguish real predators from plastic copies, and it suggests antipredator behavioral response deficit. High Zn concentrations in blood, liver, brain and skin samples are associated with deficit caused by the exposure to ZnO NPs. To the best of our knowledge, the current study is in the first to evidence that ZnO NPs induce changes in antipredator behavioral responses, even under ephemeral conditions and at low concentrations. However, the exposure to ZnO NPs can be a risk to the health of the assessed individuals and to the dynamics of their populations if the present antipredator behavioral response test results are extrapolated to the ecological context.

O tomateiro é acometido por ataque de várias doenças e pragas durante o ciclo, às quais podem ocasionar significativas perdas na quantidade e na qualidade dos frutos. Dentre as pragas e doenças de importância, destacam-se a mosca-branca, o ácaro-vermelho e o oídio. A principal forma de controle de insetos, ácaros e fungos que afetam a cultura consiste na pulverização de agrotóxicos, estratégia esta que, além de elevar sobremaneira o custo de produção, traz impactos negativos para o meio ambiente e para a saúde humana. Neste contexto, substâncias alternativas inócuas ou de baixa toxicidade, denominadas bioestimulantes agrícolas, vem sendo pesquisadas de modo a compor práticas alternativas que amenizem os efeitos nocivos oriundos do uso contínuo de moléculas tóxicas. Assim, objetivou-se avaliar o efeito da aplicação exógena de bioestimulantes, sobre enzimas e aleloquímicos relacionados à resistência e parâmetros fitiotécnicos e fisiológicos em tomateiro, na presença do ácaro-vermelho, da mosca-branca e do oídio. Utilizou-se o híbrido de tomateiro BRS-Nagai em experimento em arranjo fatorial 2x5,DIC, utilizando delineamento em blocos casualisados, onde o primeiro fator foi a presença e ausência do fungo micorrízico arbuscular Rhizophagus clarus (FMA´s) enquanto que o segundo fator foi a utilização de quatro bioestimulantes: testemunha (T), Ácido salicílico (AS), fertilizante silicatado (dióxido de silício – 68,2% + carbonato de Cálcio – 31,8%) (FS), mistura de biofertilizantes em pulverização (FSCA+SFA) e Bacillus velezensis (BV). Foram avaliados teores de proteínas totais solúveis, atividade das enzimas fenilalanina amônia-liase, catalase, peroxidase, teor de zingibereno, quantificação de tricomas, fotossíntese, transpiração, clorofila, massa fresca de frutos, altura das plantas, distância entre cachos e a infestação de adultos de mosca-branca, ácaro vermelho e do oídio. Foi observado que uso de substrato inoculado com o FMA proporcionou incremento de proteínas totais solúveis reduzindo a atividade da catalase e peroxidase. A aplicação de Bacillus velezensis aumentou os teores foliares de zingibereno. Já a mistura de biofertilizantes ou B. velezensis elevaram o índice Falker, sendo que alterações significativas não foram detectadas para transpiração e fotossíntese. FMA´s promoveram aumento da altura das plantas, bem como a distância entre cachos, estimularam aumento no número de tricomas, aumentaram o teor de zingibereno quando associados com bacilos, reduziu a população de mosca-branca em sinergismo com a mistura de bioestimulantes (FSCA + SFA). Também promoveu redução na quantidade de ácaros em associação com AS e reduziu a incidência de oídio no tratamento isolado e associado ao tratamento com mistura de biofertilizantes. The tomato is attacked by several diseases and pests during the cycle, causing damage of substantial economic importance. Among the most important pests and diseases, whitefly, red mite and powdery mildew stand out. The main way to control this key pest has been through chemicals, but with low efficiency and high cost. As an alternative, the use of viable alternative methods, one should evaluate new pest control methods. Thus, the objective was to evaluate the effect of exogenous application of biostimulants, through tomato seed treatment on enzymes and allelochemicals related to resistance, phytotechnical and physiological parameters in tomato, and on the occurrence of red mite, whitefly and powdery mildew. BRS-Nagai tomato seeds were treated using a 2x5 factorial scheme, where the first factor was the presence and absence of the arbuscular mycorrhizal fungus Rhizophagus clarus (FMA's) while the second factor was the use of five biostimulants: control (T) , Salicylic Acid (AS), Silicate Fertilizer (Silicon Dioxide – 68.2% + Calcium Carbonate – 31.8%) (FS), Biofertilizer Mixture Spray (FSCA+SFA) and Bacillus velezensis (BV). Total soluble proteins, activity of phenylalanine ammonia-lyase, catalase, peroxidase, zingiberene content, quantification of trichomes, photosynthesis, transpiration, chlorophyll, fruit mass, plant height and distance between clusters were evaluated. The experiments were The experiments were carried out in the greenhouse of the State University of Londrina, in completely randomized blocks with five replications, with tests of normality, homoscedasticity, comparison of means and Spearman correlation. It was observed that the use of substrate inoculated with FMA provided an increase in total soluble proteins and reduced catalase and peroxidase activity. The application of Bacillus velezensis increased zingiberene foliar contents. The mixture of biofertilizers or B. velezensis increased the Falker index, and significant changes were not detected for transpiration and photosynthesis. FMA´s promoted an increase in plant height, as well as the distance between clusters, stimulated an increase in the number of trichomes, increased zingiberene content when associated with bacilli, reduced the amount of whitefly in synergism with the mixture of biostimulants (FSCA + SFA) in mixture. It also promoted a reduction in the number of mites in association with AS and reduced the incidence of powdery mildew in the isolated treatment and associated with the treatment with a mixture of biofertilizers.

A soja (Glycine max (L.) Merr.) é considerada a principal oleaginosa cultivada no mundo, sendo o Brasil maior produtor e exportador dessa cultura, movimentando mais de U$ 32 bilhões na safra 2019/2020. A produção da soja no Brasil é severamente limitada pelas restrições impostas por fatores bióticos e abióticos, destacando as doenças de solo devido a forte influência na produtividade e a dificuldade na erradicação. Nesse contexto, o presente trabalho teve como objetivo avaliar o potencial da cepa CMLL06 como biocontrole para diferente patógenos de solo na cultura da soja. Nesse estudo inclui o sequenciamento bacteriano, mineração de genes responsáveis pela síntese de metabólitos secundários e a atividade antifúngica e nematicida desse isolado. Essa cepa foi obtida em amostras de solo no norte do Paraná, Brasil, e com base nas informações da identidade média de nucleotídeos e na hibridização digital DNA-DNA foi identificado como Bacillus velezensis. O genoma dessa cepa foi estimado em 3.981.409 pb com um teor de GC de 46,48% e apresentou 4047 sequências de DNA codificantes. Com base na informação do webserver antiSMASH 5.1.0, foram identificados 13 clusters de biossíntese de metabólitos secundários, no qual alguns desses tem um importante papel na supressão de patógenos e na promoção de crescimento da planta. A cepa CMLL06 apresentou atividade antifúngica in vitro (Macrophomia phaseolina, Rhizoctonia solani e Sclerotinia sclerotiorum) e in vivo (Sclerotinia sclerotiorum), com incremento de 54 e 26% na germinação e no número de plântulas sadias de soja, respectivamente, quando comparado com o controle (com infecção). Em relação a supressão de nematoides na soja, apresentou uma porcentagem de controle de 69 e 60% para Meloidogyne javanica e Pratylenchus brachyurus, respectivamente. Com base nesses resultados, essa cepa tem um importante papel para o controle de doenças do solo na cultura soja, sendo promissor para o desenvolvimento de produtos comerciais. Soybean (Glycine max (L.) Merr.) Is considered the main oilseed cultivated in worldwide, with Brazil being the largest producer and exporter of this crop, with a turnover of more than U $ 32 billion in the 2019/2020 harvest. Soybean production in Brazil is severely limited by restrictions imposed by biotic and abiotic stress, highlighting soil diseases due to the strong influence on yield and the difficulty in eradication. In this context, the present study aimed to evaluate the potential of the CMLL06 strain as a biocontrol for different soil-borne pathogens in soybean. This study includes bacterial sequencing, mining of genes responsible for the synthesis of secondary metabolites and the antifungal and nematicidal activity of this strain. This strain was obtained from soil samples in northern Paraná, Brazil, and based on information on the average nucleotide identity and on digital DNA-DNA hybridization, it was identified as Bacillus velezensis. The genome of this strain was estimated at 3,981,409 bp with a GC content of 46.48% and presented 4047 coding DNA sequences. Based on information from the antiSMASH 5.1.0 webserver, 13 secondary metabolite biosynthesis clusters were identified, in which some of these have an important role in suppressing pathogens and promoting growth plant. The strain CMLL06 showed antifungal activity in vitro (Macrophomia phaseolina, Rhizoctonia solani and Sclerotinia sclerotiorum) and in vivo (Sclerotinia sclerotiorum), with an increase of 54 and 26% in germination and in the number of healthy soybean seedlings, respectively, when compared with the control (with infection). Regarding nematode suppression in soybean, it presented a control percentage of 69 and 60% for Meloidogyne javanica and Pratylenchus brachyurus, respectively. Based on these results, this isolate plays an important role in the control of soil-borne diseases in soybean crops, being promising for the development of commercial products.

leptospirose, causada por espiroquetas do gênero Leptospira, é uma zoonose globalmente disseminada, negligenciada e emergente. O isolamento de leptospiras é o primeiro passo para sua caracterização molecular e genotipagem, no entanto, para os sorovares mais fastidiosos, a cultura e isolamento são controversos e laboriosos. Com relação ao controle e tratamento da leptospirose, muitos estudos são realizados, mas ainda não existe uma vacina eficaz contra todas as espécies patogênicas ou variedade de antimicrobianos com atuação sobre o quadro de leptospirúria. Assim o presente estudo visou primeiramente desenvolver estratégias de cultura e isolamento para as leptospiras fastidiosas e utilizar métodos rápidos de diagnóstico molecular para caracterização e tipificação dos sorovares. Posteriormente, a partir do isolamento de novas estirpes, o sequenciamento genômico foi realizado com a finalidade de obtenção de dados de genômica comparativa com outras estirpes disponíveis no GenBank para uma busca in silico de alvos drogáveis e vacinais. No primeiro estudo, três formulações de meios de cultura líquidos básicos foram produzidas para isolar e manter leptospiras. Em cada formulação (A, B e C) foram adicionados diferentes suplementos para ajudar no crescimento de leptospiras fastidiosas: piruvato de sódio, enzima superóxido dismutase e soro fetal bovino. Durante o período aproximado de 55 dias, adotou-se estratégias de troca entre as três formulações de acordo com a observação em microscopia de campo escuro e ao final desse período houve sucesso no isolamento de três estirpes do sorovar Hardjo (dois genotipos, Hardjobovis e Hardjoprajitno) com adaptação total ao meio de cultura. Posteriormente, estas culturas foram avaliadas com uso da microscopia eletrônica e notou-se diferenças na morfologia e viabilidade de acordo com a composição do meio. No segundo estudo, os genomas desses dois genotipos foram sequenciados, montados e depositados no GenBank. A partir desses dados e também de outros genomas de outras espécies de Leptospira foi possível realizar uma abordagem de com base na vacinologia reversa, genômica comparativa e de docking molecular com o uso de ferramentas de bioinformática. Para os alvos vacinais, os resultados com base nas características da probabilidade de adesão, TMHMM e densidade de epítopos, sugerem que cinco alvos são bons candidatos e podem ser testados rapidamente em novas formulações de vacinas e posteriormente testados in vivo. Para a etapa de docking molecular, oito proteínas preditas como citoplasmáticas e identificadas como essenciais para a sobrevivência de leptospiras foram consideradas bons alvos para novos medicamentos. Destacando a proteína de divisão celular FtsZ, que foi o alvo identificado com as melhores características de ligação e, por meio de uma triagem virtual, o ZINC04259719 foi identificado como a melhor molécula para ligar a essa proteína, a fim de inibir sua função eliminando o patógeno. A vacinologia reversa e o docking molecular são poderosas ferramentas de bioinformáticas que possibilitam uma busca rápida e sem procedimentos laboriosos, por alvos proteicos específicos que possam ser candidatos a uma nova vacina ou medicamento no controle da leptospirose. Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonosis. The leptospires isolation is the first step towards its molecular characterization and genotyping, however, for the most fastidious serovars, the culture and isolation are controversial and laborious. Regarding the control and treatment of leptospirosis, many studies have been carried out, but there is not yet an efficient vaccine against all pathogenic species or more variety of antimicrobials acting on leptospiruria. Thus, the present study aimed primarily to develop culture and isolation strategies for fastidious leptospires and using rapid molecular diagnostic methods for characterization and typing of serovars. Subsequently, from the isolation of new strains, genomic sequencing was carried out in order to obtain comparative genomic data with other strains available at GenBank for an in silico search for drug and vaccine targets. In the first study, three formulations of basic liquid culture media were produced to isolate and maintain leptospires. In each formulation (A, B and C) different supplements were added to help the growth of fastidious leptospires: sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum. During the approximate period of 55 days, exchange strategies were adopted between the three formulations according to the observation in dark field microscopy and at the end of this period, there was success in the isolation of three strains of serovar Hardjo (two genotypes, Hardjobovis and Hardjoprajitno) with total adaptation to the culture medium. Subsequently, these culture was evaluated through electron microscopy and differences in morphology and viability were noted according to the composition of the medium. In the second study, the genomes of these two genotypes were sequenced, assembled and deposited on GenBank. From these data and also from other genomes of Leptospira species, it was possible to carry out an approach based on reverse vaccinology, genomic comparative and molecular docking with the use of bioinformatics tools. For vaccine targets, results based on characteristics of probability of adherence, TMHMM and epitope density, suggest that five targets are good candidates and can be tested quickly in new vaccine formulations and subsequently tested in vivo. For the molecular docking step, eight proteins predicted as cytoplasmic and considered essential for the survival of leptospires were considered good targets for new drugs. Highlighting the cell division protein FtsZ, which was the target identified with the best binding characteristics and through a virtual screening the ZINC04259719 was identified as the best molecule for linked to this protein in order to inhibit its function and consequently eliminating the microorganism. Reverse vaccinology and molecular docking are powerful bioinformatics tools that enable a quick discovery, without laborious procedures, for specific protein targets that may be candidates for a new vaccine or therapeutic target to leptospirosis control.

O cogumelo Agaricus subrufescens é mundialmente conhecido devido a presença de algumas substâncias bioativas e ao seu alto valor nutricional que pode ser potencializado com a adição de alguns tipos de suplementos. Desta forma o objetivo deste trabalho foi determinar a composição química, atividade antioxidante e fenóis totais das três diferentes linhagens do cogumelo A. subrufescens [originárias do Japão (JP), Brasil (ABZ) e Bélgica (T2)] cultivadas com diferentes tipos de suplementos artesanais (com e sem adição de formol) produzidos a partir de resíduos industriais: amendoim (casca de vagem e amendoim), fruta (resíduos de acerola), combinação (farelo de soja, milho, trigo e algodão) e mistura (33,3% de cada suplemento artesanal), além de suplementos comerciais: setas e champfood. O estudo da composição química foi realizado de acordo com os métodos descritos pela Associação Oficial de Métodos Analíticos (AOAC) e o perfil cromatográfico dos extratos foram analisados por cromatografia líquida de alta eficiência (CLAE-UV/DAD). Para o tratamento de dados foi utilizada a técnica quimiométrica de análise de fatores (AF). A capacidade antioxidante dos extratos foi avaliada pelo método de DPPH•, do poder de redução do ferro (FRAP) e pela capacidade de absorção do radical oxigênio (ORAC), e o teste de fenóis totais foi realizado pelo método de Folin-Ciocalteu. A proposta de identificação das substâncias foi feita utilizando a cromatografia líquida acoplada a espectrometria de massas (CL-EM/EM). A AF da composição química demonstrou que as linhagens ABZ e T2 possuem maiores correlações com os teores de fibras e proteínas em comparação às amostras da linhagem JP. Já em relação ao perfil cromatográfico do extrato acetato de etila a AF demonstrou que os cogumelos suplementados com mistura formol e mistura apresentaram semelhança entre as amostras cultivadas com os suplementos comerciais champfood e setas para as três linhagens. Para a capacidade redutora do DPPH• o extrato mais ativo foi apresentado pela linhagem T2 suplementada pelo suplemento fruta formol (55,89 ± 2,06 mg EAG/100g); pelos métodos FRAP e ORAC os mais potentes resultados foram obtidos para linhagem ABZ com a suplementação do comercial setas (242,86 ± 37,40 mg EAG/100g e 5,95 ± 0,58 g EAG/100g, respectivamente), estaticamente iguais aos valores obtidos com o suplemento artesanal mistura (155,02 ± 25,00 mg EAG \1 00g e 5,40 ± 0,36 g EAG \1 00g). A linhagem ABZ também apresentou o maior teor de compostos fenólicos totais. Através da análise de CL-EM/EM do extrato hidroalcoólico foi possível a identificação de cinco substâncias, dentre as quais destacou-se o ácido p-cumárico por possuir atividade antioxidante relatada na literatura. Já em relação ao extrato acetato de etila foram identificadas sete substâncias, sendo que a esculetina relatada pela primeira vez no cogumelo A. subrufescens. Assim, verificou-se que os suplementos artesanais são uma alternativa viável aos comerciais e que o tipo de suplementação influencia significativamente na composição final das diferentes linhagens de A. subrufescens estudadas, sendo que, a linhagem ABZ destacou-se por apresentar resultados promissores com o suplemento artesanal mistura. The mushroom Agaricus subrufescens is known worldwide due to the presence of some bioactive compounds and its high nutritional value that can be enhanced with the addition of some types of supplements. The aim of this work was to determine the chemical composition, antioxidant activity and total phenols of the three different strains of the A. subrufescens mushroom [from Japan (JP), Brazil (ABZ) and Belgium (T2)] cultivated with different types of supplements (with and without added formaldehyde), produced from industrial waste: peanut (grain or nut and peanut), fruit (acerola residues), combination (soybean meal, corn, wheat and cotton) and blend (33.3% of each handmade supplement), in addition to commercial supplements: setas and champfood. The study of chemical composition was performed according to the methods described by the Official Association of Analytical Methods (AOAC) and the chromatographic profile of the extracts were analyzed by high performance liquid chromatography (HPLC-UV/DAD). For the data treatment, the chemical factor analysis (FA) technique was applied. The antioxidant capacity of the extracts was evaluated by the DPPH method, ferric reducing anti- oxidant power (FRAP) and the oxygen radical absorption capacity (ORAC), and the total phenol test was performed by the Folin-Ciocalteu method. The compounds identification proposal was carried out using liquid chromatography coupled to mass spectrometry (LC-MS/MS). The FA of the chemical composition showed that the ABZ and T2 strains have higher correlations with the fiber and protein contents compared to the sample JP strain. In relation to the chromatographic profile of the ethyl acetate extract, the FA showed that the mushrooms supplemented with formaldehyde mixture and mixture presented similarity between the samples cultured with the commercial supplements champfood and setas for the three strains. For the scavenging ability of DPPH• the most active extract was presented by the T2 strain supplemented with the formalin supplement (55.89 ± 2.06 mg GAE / 100g), by the FRAP and ORAC assays the most potencies were developed for the ABZ strain with commercial supplementation Setas (242.86 ± 37.40 mg GAE / 100 g and 5.95 ± 0.58 g GAE / 100 g, respectively) were statistically the same as those obtained by the FRAP and ORAC methods, (155.02 ± 25.00 mg EAG \1 00g and 5.40 ± 0.36 g GAE \1 00g). The ABZ strain also showed the highest total phenolic compounds content. Through the analysis by LC-MS/MS of the hydroalcoholic extract it was possible to identify five compounds, among which the p-coumaric acid was outstanding because it has antioxidant activity reported in the literature. In relation to the ethyl acetate extract, seven compounds were identified, and esculetin was reported in the A. subrufescens mushroom for the first time. Thus, it was verified that the handmade supplements are a viable alternative to the commercial ones and the type of supplementation influences significantly in the final composition of the different strains of A. subrufescens studied, being that the ABZ strain highlighted to present promising results with the handmade supplement blend.

Proteus mirabilis é um dos principais agentes causadores de infecções urinárias associadas à catéter. Os tratamentos contra infecções bacterianas têm se tornado cada vez mais ineficazes devido à rápida emergência de bactérias resistentes a antimicrobianos, levado à necessidade crítica de novos agentes antimicrobianos. Nanopartículas de prata (AgNPs) aparecem como candidatas por conta de sua atividade antimicrobiana e baixa toxicidade aos humanos. Este trabalho teve como objetivo verificar a atividade de AgNPs biossintetizadas pelo fungo Fusarium oxysporum contra isolado clínico de P. mirabilis uropatogênico e verificar a atividade antimicrobiana de catéteres de Foley revestidos com essas AgNPs. As AgNPs foram sintetizadas pela adição de AgNO3 ao filtrado fúngico. Análise de tamanho, potencial Zeta e morfologia das AgNPs foram determinados por espalhamento de luz dinâmico, laser doppler eletroforese, e microscopia eletrônica de transmissão, respectivamente. Os ensaios de microdiluição em caldo e cinética de tempo de morte foram usados para determinar a atividade antibacteriana das AgNPs. O ensaio de redução do XTT foi utilizado para determinação da atividade antibiofilme. Além disso, fragmentos de catéter foram revestidos com duas e três camadas de AgNPs usando polidopamina como agente ancorante. A caracterização da superfície foi realizada EDX. Pedaços de catéter revestido foram incubados em urina inoculada com P. mirabilis e o tempo necessário para incrustação foi determinado. Imagens de MCE foram realizadas para comparar a formação de biofilme entre os catéteres pristino e revestidos. As AgNPs apresentaram formato esférico, com tamanho médio de 126,3 nm e potencial Zeta de -36,86 mV. A CIM foi 62,5 µM, causando inviabilidade celular total após 4 horas de ação. A formação de biofilme foi inibida em 76,4% e o biofilme maduro foi reduzido em 1,9%. A análise por EDX confirmou a presença de prata. A incrustação dos catéteres de 2 e 3 camadas aconteceu após 13 e 20 dias, respectivamente, enquanto o catéter pristino apresentou incrustação no primeiro dia. MCE mostrou que o revestimento do catéter reduziu a intensidade de fluorescência em mais de 98%. Dessa forma, conclui-se que AgNPs biossintetizadas por F. oxysporum possuem grande atividade antibacteriana e antibiofilme, sendo uma potencial alternativa ao tratamento com antibióticos em infecções por P. mirabilis uropatogênico. Proteus mirabilis is one of the main causative agent of catheter associated urinary tract infection. Treatment of bacterial infections are becoming increasingly inefficient due to the rapid emergence of resistant bacteria, leading to a critical need for new antimicrobial agents. Silver nanoparticles (AgNPs) are promising candidates by it antibacterial activity and low toxicity for humans. This work intended to assess the activity of AgNPs biosynthesized by Fusarium oxysporum against a clinical isolate of uropathogenic P. mirabilis and assess the antimicrobial activity of Foley catheter coated with those AgNPs. AgNPs were synthesized by adding AgNO3 to fungal filtrate. AgNPs analisys of size, Zeta potential and morpholgy were determined by dynamic light scattering, laser doppler electrophoresis and transmition electromicroscopy, respectively. Broth microdilution and time-kill kinetics assays were used to determine the antibacterial activity of the AgNPs. The XTT reduction assay was employed to determine the antibiofilm effect. Beyond that, fragments of catheter were coated with two and three AgNPs layers using polydopamine as the surface anchor. Thickness and homogeneity were confirmed by EDX and SEM. Pieces of coated catheter were incubated in urine inoculated with P. mirabilis and the time taken for encrustation to occur was recorded. SCM images were taken to compare biofilm formation between pristine and coated catheters. AgNPs showed spherical shape, an average size of 126,3 nm e Zeta potential of -36,86 mV. MIC was found to be 62,5 µM, causing total loss of viability after 4 hours of action. Biofilm formation was 76,4% lower and mature biofilm was reduced by 1,9%. EDX analisys confirmed presence and homogeneity of AgNPs on coating. Encrustation of the two and three AgNPs layers catheters occurred after 13 and 20 days, respectively, while pristine catheter was encrusted on the first day. SCM showed that the coating on catheter was able to reduce the flourescence intensity by more than 98%. Therefore, it can be concluded that the AgNPs biosynthesized by F. oxysporum has great antibacterial and antibiofilim activity, being considered as a potential alternative to antibiotic treatment of infections caused by uropathogenic P. mirabilis.

O fósforo é um dos nutrientes essenciais mais exigidos durante o ciclo de vida vegetal, considerado um dos mais limitantes na produção agrícola. O desenvolvimento de tecnologias que contribuam para um melhor aproveitamento do P do solo, gerando menor demanda por fertilizantes químicos é indispensável uma vez que se trata de um nutriente esgotável e não-renovável. Fungos Micorrízico Arbusculares (FMA) são conhecidos por estabelecerem associações simbióticas com raízes de plantas, auxiliando na absorção de nutrientes, principalmente do P. Neste contexto, a formulação de um inoculante a base de um FMA, que possa ser utilizado em larga escala, é uma alternativa para auxiliar na redução das quantidades de fertilizantes fosfatados utilizados. O objetivo deste trabalho foi avaliar a eficiência da vermiculita, da turfa e do fosfato de rocha, como veículos na inoculação de sementes de soja (Glycine max) e milho (Zea mays) para o FMA Rhizophagus clarus produzido in vitro. O experimento foi conduzido em área experimental da Universidade Estadual de Londrina, Brasil. Foram elaborados sete tratamentos, distribuídos em cinco blocos casualizados, sendo: Tratamento Agronômico (TA); MA + Turfa + P (MAT+P); MA + Vermiculita + P (MAV+P); MA + Fosfato de Rocha + P (MAFR+P); MA + Turfa - P (MAT-P); MA + Vermiculita - P (MAV-P); MA + Fosfato de Rocha - P (MAFR-P). As plantas foram avaliadas quanto a: Colonização micorrízica; matéria seca; P e N da parte aérea e produtividade de grãos. Para soja, foi observado incremento na produtividade de até 16,3% em relação a TA, com a utilização de vermiculita como veículo para o inóculo, sem adubação fosfatada. Já as plantas de milho, utilizando turfa como veículo e adubação fosfatada, obtiveram ganhos de até 20% em produtividade. Phosphorus is one of the most essential nutrients required during the cycle of plant life, considered one of the most limiting agricultural production. The development of technologies that contribute to a better use of soil P, generating less demand for chemical fertilizers is essential, since it is an exhaustible nutrient and non-renewable. Arbuscular mycorrhizal fungi (AMF) are known for establishing symbiotic associations with roots of plants, aiding in the absorption of nutrients, particularly P. In this context, the formulation of an AMF inoculant, which can be used in large scale, is an alternative to help reduce the quantities of phosphate fertilizers used. The objective of this study was to evaluate the efficiency of vermiculite, peat and rock phosphate, as vehicles in soybean seed inoculation (Glycine max) and corn (Zea mays) to the AMF Rhizophagus clarus produced in vitro. The experiment was carried in experimental area of the State University of Londrina, Brazil. Seven treatments were developed, distributed in five randomized blocks being: Agronomic Treatment (AT); MA + Peat + P (MAT+P); MA + Vermiculite + P (MAV+P); MA + Rock Phosphate + P (MAFR+P); MA + Peat - P (MAT-P); MA + Vermiculite - P (MAV-P); MA + Rock Phosphate - P (MAFR-P). The plants were evaluated for; Mycorrhizal colonization; dry matter; P and N shoot and grain yield. For soybean was observed to increase the productivity of 16.3% compared to AT with the use of vermiculite as the carrier for the inoculum, no phosphorus fertilization. As for the corn plants using peat as a vehicle and phosphate fertilizer, made gains of up to 20% in productivity.

O Brasil é um dos países com maior produtividade agrícola do mundo. Entretanto, o cultivo no país sofre ação de agentes patógenicos que prejudicam a qualidade da colheita. Dentre eles, estão fungos fitopatógenos do gênero Aspergillus, que agem sobre os produtos no pós colheita, armazenados em silos, moinhos, locais de manipulação e estocagem na pré-venda. A principal implicação destes organismos é a produção de micotoxinas, metabólitos tóxicos a seres humanos e outros animais. As toxinas muitas vezes não alteram as características físicas ou organolépticas dos alimentos, mas sempre apresentam potencial de dano. As micotoxinas podem ser carcinogênicas, mutagênicas, hepato e nefrotóxicas e podem causar efeitos imunossupressores, entre outros problemas. Para conter esta produção é importante impedir a germinação dos esporos, o desenvolvimento de hifas (crescimento miceliano). Sendo assim, o intuito desta pesquisa foi a utilização de compostos biogênicos e semi-sintéticos contra fungos toxigênicos: nanopartícula de prata e a sinvastatina, respectivamente. A sinvastatina é uma estatina empregada na terapêutica hipolipemiante. Atua como competidora do sítio ativo da enzima HMG-CoA redutase, envolvida na produção de colesterol em mamíferos e na produção de ergosterol nos fungos. As nanoparticulas de prata agem em diversos sítios celulares em microrganismos, como na parede celular, grupos fosfato do DNA, citocromos mitocondriais, grupamentos sulfidrila e outras regiões. Testes inicias como disco e poço difusão em ágar demonstraram a sensibilidade de cinco cepas do gênero Aspergillus, sendo esses testes corroborados com a concentração inibitória mínima (CIM) demonstrando sensibilidade dos organismos. Quando os compostos foram utilizados em conjunto a concentração inibitória decaiu entre duas e quatro vezes daquelas observadas na CIM, demonstrando poder sinérgico e aditivo. Testes complementares como concentração fungicida mínima demonstraram que estes compostos são fungicidas entre quatro e oito concentrações superiores a observadas na CIM.Estes compostos também foram capazes inibir a formação de biofilme em placas. Por fim, através de microscopia eletrônica de varredura, pudemos observar mudanças estruturais na forma vegetativa do fungo (hifas truncadas e com perda da estrutura formal) e inibição da germinação dos esporos ou germinação anormal. Estes resultados mostram que nanopartículas de prata e sinvastatina, isoladas e principalmente conjugadas, atuam eficientemente contra fungos toxigênicos, indicando um potencial uso destas drogas em etapas diversas da cadeia de produção alimentar. Brazil is one of the countries with higher agricultural productivity in the world. However, cultivation in the country suffers action of pathogens that damage crop quality. Among them are phytopathogenic fungi of the Aspergillusgenus, acting on the products also in post-harvest, stored in silos, mills, handling and storage sites in pre-sales. The main implication of these organisms is the production of mycotoxins, metabolites toxic to animals and humans. These secondary metabolites often do not alter the physical or organoleptic characteristics of the food, but always have potential for harm. Mycotoxins can be carcinogenic, mutagenic, hepato and nephrotoxic and can cause immunosuppressive effects, among other problems. To counter this production is important to prevent germination of the spores, hyphal development (mycelium growth). Thus, the aim of this research is to use natural and semi-synthetic compounds against toxigenic fungi: Silver nanoparticles and simvastatin, respectively. Simvastatin is a statin used in the lipid-lowering therapy. Acts as a competitor of the enzyme HMG-CoA reductase, which is involved in the production of cholesterol in mammals and in the production of ergosterol in fungi. The silver nanoparticles act on many cell sites in microorganisms such as cell wall, DNA phosphate groups, mitochondrial cytochromes, sulfhydryl groups and other regions. Preliminary tests with disc or well diffusion demonstrated the sensitivity of five strains of the genus Aspergillus, which was corroborated by other method, MIC (minimum inhibitory concentration). By this means, sensitivity of fungi to the coumpounds was confirmed. When the compounds are used in conjunction inhibitory concentration has fallen between two and four times regarding values found in MIC, demonstrating synergistic or additive effect. Additional tests as MFC (minimum fungicidal concentration) demonstrated that these compounds are fungicidal between five and ten concentrations greater than observed in CIM. The compounds were also able to reduce or inhibit biofilm formation on polystyrene plates. Finally, by scanning electron microscopy (SEM), we observed structural changes in the vegetative form of the fungus (truncated and loss of formal structure hyphae) and inhibition of germination of spores or abnormal germination. Moreover, it was noted that the test compounds affected the production of extracellular matrix produced by the fungi. These results show that silver nanoparticles and simvastatin, isolated or conjugated, act effectively against toxigenic fungi, indicating a potential use of these drugs in various stages of the food production chain.

O Huanglongbing (HLB, ex-greening),é considerado a mais grave das doenças de citros. Não há medidas de controle efetivas e de baixo custo, nem mesmo métodos curativos para o HLB. Neste sentido, este estudo teve como objetivo avaliar a atividade da fração F4A resultante do metabolismo secundário de Pseudomonas aeruginosa (cepa LV) em plantas de Citrus sinensis no controle do Huanglongbing. O primeiro experimento teve como objetivo avaliar diferenças na população bacteriana de Ca. Liberibacter asiaticus pela técnica de q-PCR, plantas de Citrus sienensis do cultivar Valencia foram inoculados com Ca. Liberibacter asiaticus pela técnica de enxertia e três concentrações da fração F4A foram avaliadas sendo elas 10µg/mL-1 100µg/mL-1 e 1000µg/mL-1 em dois tempos de aplicação denominados de preventivo e curativo, tendo como delineamento experimental blocos cazualizados, com sete replicas biológicas para cada tratamento. O segundo experimento teve como objetivo investigar o potencial indutor de resistência da fração através da expressão do gene β-1,3 glucanase em plantas de Citrus sinensis (Valencia) pela técnica de RT-PCR. Três replicatas biológicas foram utilizadas por tratamento, os tratamentos foram os seguintes: aplicação da fração F4A nas concentrações de 10µg/mL-1 100µg/mL-1 e 1000µg/mL-1, como controle positivo foi utilizado o produto comercial BION e como controle negativo apenas água destilada, o experimento foi avaliado 24h, 7 dias e 15 dias após a aplicação dos compostos. No primeiro experimento o tratamento curativo com 100µg/mL-1 da Fração F4A foi capaz de reduzir em duas unidade de Log a população de Ca. Liberibacter asiaticus em comparação com as plantas testemunhas. No segundo experimento a expressão do gene de resistência para β-1,3 gluacanase foi 9 vezes mais expressa no tratamento com 100µg/mL-1 da Fração F4A após 24h da aplicação e 12,9 vezes mais expressa após o sétimo dia de aplicação. Os resultados deste estudo mostram-se promissores e sugerem que os compostos produzidos pela cepa LV de Pseudomonas aeruginosa surgem como uma possível alternativa no controle da doença Huanglongbing. The Huanglongbing (HLB) is considered the most serious citrus diseases. There is no effective control measures and low cost, even curative methods for HLB. Thus, this study aimed to evaluate the activity of the fraction F4A resulting from secondary metabolism of Pseudomonas aeruginosa (strain LV) in Citrus sinensis in control of HLB. Two experiments were conducted, the first plants of Citrus sinensis cultivar Valencia were inoculated with Ca. Liberibacter asiaticus by the technique of grafting and the fraction F4A three concentrations were applied, namely: 10 µg mL-1, 100 μg mL-1 and 1000 µg mL-1 and as negative control only distilled water was used. Two time intervals were evaluated termed preventive and curative, the experimental design was completely cazualizado with seven biological replicates for each treatment. After four and eight months after inoculation DNA samples were extracted and analyzed by Q-PCR technique for the quantification of Ca. Liberibacter asiaticus on plants. In the second experiment, three biological replicates of Citrus sinensis (Valencia) were used, the treatments were: for the ratio F4A concentrations of 10 µg mL-1 100 µg mL-1 and 1000 µg mL-1 as a positive control the commercial product was used BION as a negative control and only distilled water, the experiment was evaluated 24h, 7 days and 15 days after application of the compounds, and evaluated by RT-PCR to investigate the potential to induce resistance Fraction F4A, through expression of gene β-1,3 glucanase. In the first experiment curative treatment with 100 μg mL-1 fraction F4A was able to reduce by two unit Log population of Ca. Liberibacter asiaticus compared with control plants. In the second experiment the expression of the resistance gene β-1,3 glucanase was 9 times higher in treatment with 100μg mL-1 fraction F4A 24h after application and 12.9 times more expressed after the seventh day of application. The results of this study are promising and suggest that the compounds produced by LV strain of P. aeruginosa emerge as a possible alternative for disease control HLB.

O tomate é uma hortaliça de grande relevância econômica mundial e nacional. A podridão mole do tomateiro é causada pela bactéria Gram negativa Pectobacterium carotovorum subsp. carotovorum (Pcc) que coloniza os vasos condutores da planta em qualquer fase do cultivo, esta doença não é controlada com produtos químicos, existindo a necessidade de novas alternativas para controle da doença. O uso de Pseudomonas sp. tem potencial para exploração como pesticida biológico, reduzindo o uso de pesticidas químicos na agricultura. Este estudo teve como objetivo avaliar a ação de compostos extracelulares de Pseudomonas aeruginosa no controle da Pcc em tomateiro. O processo de produção e extração do composto antibiótico foi realizado de acordo com a patente registrada PI0803350-1 – INPI 12/09/2008.As frações antibióticas foram obtidas por cromatografia líquida em coluna a partir da purificação dos metabólitos brutos da cepa LV. Para purificação de fenazina-1-carboxiamida foi utilizada a metodologia de Shanmugaiah et al. (2010) adaptada para uso em sistema automatizado de cromatografia flash (FP 971 - Agilent®). Para avaliação da atividade antibiótica foram realizados os testes de difusão em disco, microdiluição em caldo, determinação da concentração inibitória mínima (CIM) em placa de 96 poços, avaliação ultra-estrutural in vitro por microscopia eletrônica de varredura (MEV). Na avaliação do controle da doença e da indução de resistência foram realizados testes in vivo em casa de vegetação com a F4A e o composto fenazina-1-carboxiamida, sendo avaliado também o efeito na produção do fruto. A comprovação da indução de resistência foi avaliada pela atividade de três enzimas relacionadas com o sistema de defesa das plantas: peroxidase, fenoloxidase e fenilalanina-amônia-liase, em plantas inoculadas e não, tratadas com o composto fenazina-1-carboxamida. Os testes em disco indicaram melhor atividade para a fração F4A em todas as concentrações testadas, sendo sua CIM 7.812 μg mL-1 para Pcc. O composto fenazina-1-carboxiamida não apresentou inibição da Pcc. Na MEV foram observadas mudanças morfológicas provocadas pela fração F4A na célula bacteriana após 3 hrs de incubação. Nos testes in vivo foi observado o controle da doença nas plantas tratadas, além do estímulo no crescimento e produção, mostrando uma possível indução de resistência sugerida pelo aumento das atividades enzimática. Levando-se em conta a presença do composto fenazina-1-carboxamida na fração F4A, necessita-se de mais testes para comprovação da origem da ação dos resultados obtidos. O biocontrole da Pcc com a utilização de compostos do metabolismo secundário bacteriano da cepa LV de P. aeruginosa pode ser considerado uma ferramenta no manejo deste fitopatógeno. Tomato is an important vegetable crop of major economic importance worldwide and in Brazil, present in various agricultural regions. The soft rot of tomato is caused by Gram-negative bacteria Pectobacterium carotovorum subsp. carotovorum (Pcc) that colonizes the conducting vessels of the plant at any stage of cultivation, this disease is not controlled with chemicals. The use of Pseudomonas sp. has high potential for exploitation as biological pesticide, reducing the use of chemical pesticides in agriculture there is a need for new alternatives to control the disease. The aim of this study was evaluate the action of extracellular compounds of Pseudomonas aeruginosa in the control of Pcc in tomato. The process for production and extraction of the antibiotic compound was performed according to the registered patent PI0803350-1 - PTO 12/09/2008. The antibiotic fractions were obtained by liquid vaccum column chromatography from the purification of raw metabolites of strain LV. For purification of phenazine-1-carboxamide was used methodology of Shanmugaiah et al, 2010, adaptaded for use in automated flash chromatography (PF 971 - Agilent ®). To evaluate the antibiotic activity was used testing disk diffusion broth microdilution for determining the minimum inhibitory concentration (MIC) in 96-well plate, ultrastructural evaluation in vitro by scanning (SEM) electron microscopy were performed. For evaluation of disease control and induction of resistance, in vivo tests were conducted in a greenhouse with F4A compound and phenazine-1-carboxamide, and evaluated the effect in stimulating the production of fruit. To prove the induction of resistance was evaluated the activity of three enzymes of protecting plants: peroxidase, phenol oxidase and phenylalanine ammonia-lyase in inoculated and not treated with phenazine-1-carboxamide compound. As a result, the disk tests showed the best activity for F4A fraction at all concentrations tested, with a MIC 7.812 mg mL-1 for Pcc. The compound phenazine-1-carboxamide showed no inhibition of Pcc. In MEV, morphological changes were observed in the bacterial cell after 3hrs of incubation. In vivo testing of disease control, was observed in treated plants, in addition to stimulating the growth and production, the induction of resistance suggested by the increasing enzymatic activities. Taking into account the presence of the compound phenazine-1-carboxamide fraction F4A, will be need more tests to prove the origin of the action results. The biocontrol against Pcc with compounds derived from bacterial secondary metabolism of LV strain of P. aeruginosacan be considered a tool in the management of this pathogen.

Mais da metade das infecções relacionadas à assistência à saúde são causadas por microrganismos resistentes aos antimicrobianos, desafiando a terapia com os fármacos disponíveis. A resistência bacteriana aos carbapenêmicos, com destaque para infecções causadas por Klebsiella pneumoniae produtora da arbapenemase KPC, estão associadas à elevadas taxas de mortalidade diretamente relacionadas às limitações impostas por este importante mecanismo de resistência. Em contrapartida, o desenvolvimento de novos fármacos não mantém o mesmo rítmo dos múltiplos mecanismos de resistência desenvolvidos pelas bactérias. Tendo em vista este cenário, a pesquisa por novas substâncias antimicrobianas é indispensável para o controle das infecções multirresistentes. Os produtos naturais desempenham papel importante na descoberta de novos fármacos antimicrobianos. Neste sentido, metabólitos secundários de Pseudomonas aeruginosa possuem potencial na produção de substâncias antibióticas, tendo em vista sua complexidade genética e diversidade metabólica. More than a half of healthcare associated infections cases are caused by multidrugresistant organisms, restricting the uses of antimicrobials available. Bacterial resistance to carbapenems, especially infections caused by Klebsiella pneumoniae carbapenemase KPC-producing, are associated with high rates of mortality directly related to the limitations imposed by this important resistance mechanisms. On the other hand, the development of new drugs does not keep the same of the multiple resistance mechanisms developed by bacteria. In this way, it is clear the urgent need of new and more efficacious compounds for specific treatment of infections caused by multidrug-resistant organisms. Natural products play an important role in the discovery of new antimicrobial drugs. The secondary metabolites of Pseudomonas aeruginosa has potential on producing antibiotic substance, considering your genetic complexity and metabolic diversity.

O biocombustível etanol é uma alternativa viável à utilização de combustíveis fósseis, principalmente quanto às questões ambientais, pois o etanol é uma fonte energética renovável, e quando comparado a combustíveis como o petróleo apresenta menores taxas de emissão de gases estufa. A produção de etanol é influenciada pela contaminação bacteriana durante o processo fermentativo realizado pela levedura Saccharomyces cerevisiae. As bactérias contaminantes na indústria sucroalcooleira são geralmente Gram-positivas, principalmente as espécies de Lactobacillus. O controle destes contaminantes geralmente é realizado com o uso de antibióticos, porém devido à seleção de cepas resistentes é necessária a busca de alternativas para este controle. Os produtos naturais como os metabólitos secundários bacterianos são fontes promissoras na prospecção por novos antibióticos. Neste contexto, o objetivo deste trabalho foi avaliar a atividade antibiótica das frações semi-purificadas de metabólitos produzidos por Pseudomonas aeruginosa (cepa LV) no controle de Lactobacillus sp., contaminante do processo de fermentação etanólica. Os metabólitos produzidos pela cepa LV, foram purificados por métodos cromatográficos. Todas as frações obtidas foram testadas quanto a atividade antibiótica. As Frações F6a, F6a.4, F6a.6, F6a.7 e F6a.8 e principalmente F6a.7.4, F6a.7.5 apresentaram resultados satisfatórios no controle do contaminante Lactobacillus sp., sem prejudicarem o crescimento da levedura S. cerevisiae, responsável pela produção do etanol. A concentração inibitória mínima para as frações F6a.7.4 e F6a.7.5 foi de 31, 25 µg.mL-1 e de 250 µg.mL-1 para F6a.7.6. Os resultados observados neste estudo demonstraram que compostos isolados a partir de metabólitos secundário de P. aeruginosa, cepa LV, podem ser uma ferramenta para o controle de Lactobacillus sp., importante contaminante na produção de etanol. The ethanol biofuel is a viable alternative to fossil fuel use, especially on environmental issues, as ethanol is a renewable energy source and when compared to fuels such as oil, has lower rates of greenhouse gas emissions. Ethanol production is influenced by the bacterial contamination during the fermentation process performed by the yeast Saccharomyces cerevisiae. The bacteria contaminants in the alcohol industry are generally Gram-positive, particularly the species of Lactobacillus. The control of these contaminants is usually accomplished with the use of antibiotics, however due to the selection of resistant strains is necessary to seek alternatives to this control. Natural products such as bacterial secondary metabolites are promising sources in prospecting for new antibiotics. In this context, the objective of this study was to evaluate the antimicrobial activity of semi-purified fractions of metabolites produced by Pseudomonas aeruginosa (strain LV) in control of Lactobacillus sp., Contaminating the ethanol fermentation processes. The metabolites produced by strain VL, were purified by chromatographic methods. All the fractions were tested for antibiotic activity. Fractions F6a, F6a.4, F6a.6, F6a.7 and F6a.8 and especially F6a.7.4, F6a.7.5 showed satisfactory results in controlling contaminant Lactobacillus sp., Without harming the growth of the yeast S. cerevisiae, responsible for the production of ethanol. The minimal inhibitory concentration for fractions F6a.7.4 and F6a.7.5 was 31, 25 µg.mL-1 and 250 µg.mL-1 for F6a.7.6. The results of this study demonstrate that compounds isolated from secondary metabolites of P. aeruginosa strain LV, can be na tool for controlling Lactobacillus sp. important contaminant in ethanol production.