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Molecular biology techniques allowed access to non-culturable microorganisms, while studies using analytical chemistry, as Liquid Chromatography and Tandem Mass Spectrometry, showed the existence of a complex communication system among bacteria, signaled by quorum sensing molecules. These approaches also allowed the understanding of dysbiosis, in which imbalances in the microbiome diversity, caused by antibiotics, environmental toxins and processed foods, lead to the constitution of different diseases, as cancer. Colorectal cancer, for example, can originate by a dysbiosis configuration, which leads to biofilm formation, production of toxic metabolites, DNA damage in intestinal epithelial cells through the secretion of genotoxins, and epigenetic regulation of oncogenes. However, probiotic strains can also act in epigenetic processes, and so be use for recovering important intestinal functions and controlling dysbiosis and cancer mitigation through the metabolism of drugs used in chemotherapy, controlling the proliferation of cancer cells, improving the immune response of the host, regulation of cell differentiation and apoptosis, among others. There are still gaps in studies on the effectiveness of the use of probiotics, therefore omics and analytical chemistry are important approaches to understand the role of bacterial communication, formation of biofilms, and the effects of probiotics and microbiome on chemotherapy. The use of probiotics, prebiotics, synbiotics, and metabiotics should be considered as a complement to other more invasive and hazard therapies, such chemotherapy, surgery, and radiotherapy. The study of potential bacteria for cancer treatment, as the next-generation probiotics and Live Biotherapeutic Products, can have a controlling action in epigenetic processes, enabling the use of these bacteria for the mitigation of specific diseases through changes in the regulation of genes of microbiome and host. Thus, it is possible that a path of medicine in the times to come will be more patient-specific treatments, depending on the environmental, genetic, epigenetic and microbiome characteristics of the host.

Sclerotinia sclerotiorum is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of S. sclerotiorum can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of S. sclerotiorum (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of S. sclerotiorum and assist in pathogen management and control.

Excessive use of herbicides decreases soil biodiversity and fertility. The literature on the xenobiotic response by microorganisms is focused on herbicide biodegradation as a selective event. Non-degradation systems independent of selection could allow the survival of tolerant bacteria in contaminated environments, impacting xenobiotic turnover and, consequently, bioremediation strategies. However, it is uncertain whether the response based on these systems requires selective pressure to be effective. The objective here was to analyze non-degradation phenotypes, enzymatic and structural response systems, of

Herbicides are widely used in agricultural practices for preventing the proliferation of weeds that compete with crops for survival. Upon reaching soil and water, herbicides can damage nontarget organisms, such as bacteria, which need an efficient defense mechanism to tolerate the stress induced by herbicides. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide that exerts increased oxidative stress among bacterial communities that consequently witness an increased toxicity in their microenvironments. Bacterial isolates were obtained from the biofilm of water that was contaminated with 2,4-D. This biofilm originated from the tanks containing washing water from the packaging of different pesticides, including 2,4-D. Moreover, several isolates were sensitive to biofilm toxicity; however, they remained alive in the presence of 2,4-D. The Pseudomonas sp. CMA-7.3 was selected because of its tolerance against biofilm agrochemicals. Therefore, the objective of this study is to evaluate the antioxidative response system of the Pseudomonas sp. CMA-7.3. This study also analyzed poorly evaluated enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase GPX, in the bacterial systems. The toxic effects of 2,4-D on bacteria were evaluated using mechanisms indicating oxidative stress, such as growth curve, cell viability, peroxide, and malondialdehyde. The Pseudomonas sp. CMA-7.3 was an efficient response system against the activity of antioxidant enzymes such as SOD, CAT, APX, and GPX in balancing the production of H2O2, even at high doses as 25x the field dose of the herbicide, thereby proving the toxicity of 2,4-D for this strain and showing the ability of the strain to tolerate 2,4-D. The adaptation of this microorganism to herbicide exposure is truly relevant for improving future metabolic studies on bacterial communities. The strain showed a great potential in the application and developmental prospects of a new product in the bioremediation process of environments contaminated by these herbicides.

Agriculture uses many food production chains, and herbicides participate in this process by eliminating weeds through different biochemical strategies. However, herbicides can affect non-target organisms such as bacteria, which can suffer damage if there is no efficient control of reactive oxygen species. It is not clear, according to the literature, whether the efficiency of this control needs to be selected by the presence of xenobiotics. Thus, the Pseudomonas sp. CMA 6.9 strain, collected from biofilms in an herbicide packaging washing tank, was selected for its tolerance to pesticides and analyzed for activities of different antioxidative enzymes against the herbicides Boral®, absent at the isolation site, and Heat®, present at the site; both herbicides have the same mode of action, the inhibition of the enzyme protoporphyrinogen oxidase. The strain showed tolerance to both herbicides in doses up to 45 times than those applied in agriculture. The toxicity of these herbicides, which is greater for Boral®, was assessed by means of oxidative stress indicators, growth kinetics, viability, and amounts of peroxide and malondialdehyde. However, the studied strain showed two characteristic antioxidant response systems for each herbicide: glutathione-s-transferase acting to control malondialdehyde in treatments with Boral®; and catalase, ascorbate peroxidase, and guaiacol peroxidase in the control of peroxide induced by Heat®. It is possible that this modulation of the activity of different enzymes independent of previous selection characterizes a system of metabolic plasticity that may be more general in the adaptation of microorganisms in soil and water environments subjected to chemical contaminants. This is relevant to the impact of pesticides on the diversity and abundance of microbial species as well as a promising line of metabolic studies in microbial consortia for use in bioremediation.

Herbicides have harmful effects on bacteria, despite not being their primary targets. Herbicide active molecules cause unspecific oxidative stress, affecting soil microbiome function. Microorganisms possess enzymatic and non-enzymatic oxidative stress control systems that enable them to survive in contaminated environments. It is uncertain whether these systems require specific selective pressures to remain effective against contaminants and how the toxicity is signaled. The objective of this work was to analyze the antioxidant enzymes and lipid profiles of a Pseudomonas fluorescens strain, isolated from water used in washing of pesticide containers, as response systems to the herbicide glyphosate, not present at the isolation site. The bacteria showed low growth rates, cell viability and high hydrogen peroxide quantities at 40x and 50x glyphosate concentrations used in agriculture. These findings demonstrate the toxicity of this herbicide to Pseudomonas strain. However, growth rates in the presence of up to 10x were equivalent to the control group, probably because of superoxide dismutase and the catalase isoenzyme activities that managed to maintain peroxide levels under control in the early- and mid-log phases. Identification and quantification of lipids, by gas chromatography and mass spectrometry, showed that specific fatty acids were correlated with high concentrations of glyphosate and specific growth phases in P. fluorescens , through Principal Component Analysis, suggesting a role in modulating responses to glyphosate toxicity. Nevertheless, glyphosate was not present in the isolation site and, therefore, was not previously selective for P. fluorescens . This suggest that the response system is generalist, based on physiological plasticity, indicating substantial adaptative values in environments subjected to intense pesticide contamination. A matter that emerges is what would be the consequences of a system that is able to respond to toxic substances to which soil microbiomes have not had contact on for bacterial diversity and functionality in agricultural soils.

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China’s Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients’ overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.

There is high demand for herbicides based on the necessity to increase crop production to satisfy world-wide demands. Nevertheless, there are negative impacts of herbicide use, manifesting as selection for resistant weeds, production of toxic metabolites from partial degradation of herbicides, changes in soil microbial communities and biogeochemical cycles, alterations in plant nutrition and soil fertility, and persistent environmental contamination. Some herbicides damage non-target microorganisms via directed interference with host metabolism and via oxidative stress mechanisms. For these reasons, it is necessary to identify sustainable, efficient methods to mitigate these environmental liabilities. Before the degradation process can be initiated by microbial enzymes and metabolic pathways, microorganisms need to tolerate the oxidative stresses caused by the herbicides themselves. This can be achieved via a complex system of enzymatic and non-enzymatic antioxidative stress systems. Many of these response systems are not herbicide specific, but rather triggered by a variety of substances. Collectively, these nonspecific response systems enhance the survival and fitness potential of microorganisms. Biodegradation studies and remediation approaches have relied on individually selected strains to effectively remediate herbicides in the environment. Nevertheless, it has been shown that microbial communication systems that modulate social relationships and metabolic pathways inside biofilm structures among microorganisms are complex; therefore, use of isolated strains for xenobiotic degradation needs to be enhanced using a community-based approach with biodegradation pathway integration. Bioremediation efforts can use omics-based technologies to gain a deeper understanding of the molecular complexes of bacterial communities to achieve to more efficient elimination of xenobiotics. With this knowledge, the possibility of altering microbial communities is increased to improve the potential for bioremediation without causing other environmental impacts not anticipated by simpler approaches. The understanding of microbial community dynamics in free-living microbiota and those present in complex communities and in biofilms is paramount to achieving these objectives. It is also essential that non-developed countries, which are major food producers and consumers of pesticides, have access to these techniques to achieve sustainable production, without causing impacts through unknown side effects., Environmental microbiology; Analytical chemistry; DNA analyses; Metabolic pathways; Cell communication

The wide use of pesticides in agriculture expose microbiota to stressful conditions that require the development of survival strategies. The bacterial response to many pollutants has not been elucidated in detail, as well as the evolutionary processes that occur to build adapted communities. The purpose of this study was to evaluate the bacterial population structure and adaptation strategies in planktonic and biofilm communities in limited environments, as tanks containing water used for washing herbicide containers. This biodiversity, with high percentage of nonculturable microorganisms, was characterized based on habitat and abiotic parameters using molecular and bioinformatics tools. According to water and wastewater standards, the physicochemical conditions of the tank water were inadequate for survival of the identified bacteria, which had to develop survival strategies in this hostile environment. The biodiversity decreased in the transition from planktonic to biofilm samples, indicating a possible association between genetic drift and selection of individuals that survive under stressful conditions, such as heating in water and the presence of chlorine, fluorine and agrochemicals over a six-month period. The abundance of Enterobacter, Acinetobacter and Pseudomonas in biofilms from water tanks was linked to essential processes, deduced from the genes attributed to these taxonomic units, and related to biofilm formation, structure and membrane transport, quorum sensing and xenobiotic degradation. These characteristics were randomly combined and fixed in the biofilm community. Thus, communities of biofilm bacteria obtained under these environmental conditions serve as interesting models for studying herbicide biodegradation kinetics and the prospects of consortia suitable for use in bioremediation in reservoirs containing herbicide-contaminated wastewater, as biofilters containing biofilm communities capable of degrading herbicides., Microbiology; Adaptation; Biodiversity; Bacteria; Microbial genomics; Biofilms; Microbial biotechnology; Environmental microbiology; Xenobiotics; Herbicide biodegradation; Bacterial diversity; Bioremediation; Bacterial adaptation.

Pesticides contribute to pest control and increased agricultural production; however, they are toxic to non-target organisms and they contaminate the environment. The exposure of bacteria to these substances can lead to the need for physiological and structural changes for survival, which can be determined by genes whose expression is regulated by quorum sensing (QS). However, it is not yet clear whether these processes can be induced by herbicides. Thus, the aim of this work was to determine whether there is a QS response system in a Pseudomonas fluorescens strain that is modulated by herbicides. This strain was isolated from water storage tanks used for washing pesticide packaging and was tested against herbicides containing saflufenacil, glyphosate, sulfentrazone, 2,4-D, and dicamba as active molecules. We found that this strain possibly uses QS signaling molecules to control the production of reactive oxygen species, whether those produced by the bacterium’s energy generating system or by molecules induced by the presence of saflufenacil and glyphosate. This strain used other signaling molecules for various stages of biofilm formation in the presence of herbicides containing sulfentrazone, 2,4-D, and dicamba. These findings, as an initial screening which will guide new studies, suggest that this strain has a flexibility in gene expression that allows survival in the presence of several stress-inducing molecules, regardless of previous exposure. This represents a model of metabolic and physiological plasticity. Biofilms made up of several bacterial species can use this model in agricultural environments, increasing the potential for degradation of xenobiotics, but with impacts on diversity and functionality of microbiotas in these environments.

Herbicides are continuously used to minimize the loss of crop productivity in agricultural environments. They can, however, cause damage by inhibiting the growth of microbiota via oxidative stress, due to the increased production of reactive oxygen species (ROS). Cellular responses to ROS involve the action of enzymes, including superoxide dismutase (SOD) and catalase (CAT). The objective of this study was to evaluate adaptive responses in Escherichia coli K-12 to paraquat, the active ingredient in the herbicide Gramoxone®. Mutant bacterial strains carrying deletions in genes encoding Mn-SOD (sodA) and Fe-SOD (sodB) were used and resulted in distinct levels of hydrogen peroxide production, interference in malondialdehyde, and viability. Mutations also resulted in different levels of interference with the activity of CAT isoenzymes and in the inactivation of Cu/Zn-SOD activity. These mutations may be responsible for metabolic differences among the evaluated strains, resulting in different patterns of antioxidative responses, depending on mutation background. While damage to the ΔsodB strain was minor at late log phase, the reverse was true at mid log phase for the ΔsodA strain. These results demonstrate the important role of these genes in defense against oxidative stress in different periods of growth. Furthermore, the lack of Cu/Zn-SOD activity in both mutant strains indicated that common metal cofactors likely interfere in SOD activity regulation. These results also indicate that E. coli K-12, a classical non-environmental strain, constitutes a model of phenotypic plasticity for adaptation to a redox-cycling herbicide through redundancy of different isoforms of SOD and CAT enzymes.

Molecular and bioinformatics tools for research are very important for biodiversity characterization in contaminated environments due the presence of a high percentage of nonculturable microorganisms. The wide use of pesticides in agriculture exposes microbiomes to stressful and selective conditions that demand survival strategies such as biofilm formation. The purpose of this work was to evaluate the bacterial population structure in planktonic and biofilm communities of water that was used for washing the packaging of herbicides and stored for six months in tanks. This substance is highly contaminated waste. Samples of water and biofilms from tanks and biofilms developed for short times in flasks were used for DNA isolation and 16S rRNA gene sequencing. The physicochemical conditions imposed by water used for washing the containers were inadequate for killing the bacterial genera identified according to water and wastewater standards. The variation in population structure and diversity was lower in the planktonic samples than in the biofilm samples, indicating a possible combination of genetic drift and subsequent selection of individuals surviving under stressful water conditions, such as heating and contact with agrochemicals, over a six-month period. The biofilm formation in water tanks contaminated with pesticides enabled the survival of bacterial genera harboring the essential processes for adaptation to these environments; the presence of these processes was determined according to descriptions obtained from the genomic databases. This study suggests the potential of bacterial genera identified in biofilms obtained from tanks to adapt to contaminated environments through their metabolic complexity. Thus, herbicide biodegradation kinetics can be accessed through a culturable collection obtained from these communities.

Callisto®, containing the active ingredient mesotrione (2-[4-methylsulfonyl-2-nitrobenzoyl]1,3-cyclohenanedione), is a selective herbicide that controls weeds in corn crops and is a potential environmental contaminant. The objective of this work was to evaluate enzymatic and structural changes in Pantoea ananatis, a strain isolated from water, in response to exposure to this herbicide. Despite degradation of mesotrione, probably due a glutathione-S-transferase (GST) pathway in Pantoea ananatis, this herbicide induced oxidative stress by increasing hydrogen peroxide production. Thiol fragments, eventually produced after mesotrione degradation, could be involved in increased GST activity. Nevertheless, there was no peroxidation damage related to this production, as malondialdehyde (MDA) synthesis, which is due to lipid peroxidation, was highest in the controls, followed by the mesotrione- and Callisto®-treated cultures at log growth phase. Therefore, P. ananatis can tolerate and grow in the presence of the herbicide, probably due an efficient control of oxidative stress by a polymorphic catalase system. MDA rates depend on lipid saturation due to a pattern change to a higher level of saturation. These changes are likely related to the formation of GST-mesotrione conjugates and mesotrione degradation-specific metabolites and to the presence of cytotoxic adjuvants. These features may shift lipid membrane saturation, possibly providing a protective effect to bacteria through an increase in membrane impermeability. This response system in P. ananatis provides a novel model for bacterial herbicide tolerance and adaptation in the environment. Electronic supplementary material The online version of this article (doi:10.1186/s13568-016-0240-x) contains supplementary material, which is available to authorized users.

Mesotrione is a benzoylcyclohexane-1,3-dione herbicide that inhibits 4-hydroxyphenyl pyruvate dioxygenase in target plants. Although it has been used since 2000, only a limited number of degrading microorganisms have been reported. Mesotrione-degrading bacteria were selected among strains isolated from Brazilian aquatic environments, located near corn fields treated with this herbicide. Pantoea ananatis was found to rapidly and completely degrade mesotrione. Mesotrione did not serve as a sole C, N, or S source for growth of P. ananatis, and mesotrione catabolism required glucose supplementation to minimal media. LC–MS/MS analyses indicated that mesotrione degradation produced intermediates other than 2-amino-4-methylsulfonyl benzoic acid or 4-methylsulfonyl-2-nitrobenzoic acid, two metabolites previously identified in a mesotrione-degrading Bacillus strain. Since P. ananatis rapidly degraded mesotrione, this strain might be useful for bioremediation purposes.

The aim of this study was to investigate the antioxidant responses of three bacteria (SD1, KD and K9) isolated from soil previously treated with the herbicides metolachlor and acetochlor. By 16S rRNA gene sequencing, we determined that SD1 is phylogenetically related to Enterobacter asburiae, while KD and K9 have divergent genomes that more closely resemble that of Enterobacter amnigenus. Decreased levels of lipid peroxidation were observed in SD1 and KD following treatment with 34 mM metolachlor or 62 mM acetochlor, respectively, indicating that both bacteria were able to adapt to an increase in ROS production. In the presence of 34 mM metolachlor or 62 mM acetochlor, all bacterial isolates exhibited increases in total catalase (CAT) activity (81% for SD1, 53% for KD and 59% for K9), whereas total SOD activity (assessed based on the profile and intensity of the bands) was slightly reduced when the bacteria were exposed to high concentrations of the herbicides (340 mM metolachlor or 620 mM acetochlor). This effect was due to a specific reduction in SOD IV (K9 and KD isolates) by 45% and 90%, respectively, and SOD V (SD1 isolate) isoenzymes by 60%. The most striking result was obtained in the SD1 isolate, where two novel isoenzymes of glutathione reductase (GR) that responded specifically to metolachlor were identified. In addition, acetochlor was shown to induce the expression of a new 57 kDa protein band in the K9 and KD isolates. The bacteria isolated from the herbicide-contaminated soil exhibited an efficient antioxidant system response at herbicide concentrations of up to 34 mM metolachlor or 62 mM acetochlor. These data suggest a mechanism for tolerance that may include the control of an imbalance in ROS production versus scavenging. The data suggest that specific isoenzymes of CAT and GR could be involved in this herbicide tolerance mechanism.

Biological control consists of using one organism to attack another that may cause economic damage to crops. Integrated Pest Management (IPM) is a very common strategy. The white mold produced by Sclerotiniasclerotiorum (Lib.) causes considerable damage to bean crops. This fungus is a soil inhabitant, the symptoms of which are characterized by water-soaked lesions covered by a white cottony fungal growth on the soil surface and/or the host plant. Possible biological control agents taken from plants are being investigated as phytopathogen inhibitors. These are endophytic microorganisms that inhabit the intercellular spaces of vegetal tissues and are often responsible for antimicrobial production. The objective of the present study was to select endophytic fungi isolated from comfrey (Symphytumofficinale L.) leaves with in vitro antagonist potential against the phytopathogenic fungus S. sclerotiorum. Twelve isolates of endophytic fungi and a pathogenic strain of S. sclerotiorum were used in the challenge method. With the aid of this method, four endophytes with the best antagonistic activity against S. sclerotiorum were selected. Pathogen growth inhibition zones were considered indicative of antibiosis. The percentages of pathogenic mycelia growth were measured both with and without the antagonist, resulting in growth reductions of 46.7% to 50.0% for S. sclerotiorum. These analyses were performed by evaluating the endophytic/pathogenic mycelia growth in mm/day over an eight-day period of antagonistic tests.O controle biológico consiste no uso de organismos que atacam outros que causam danos a culturas de plantas. Esta é uma estratégia muito utilizada no Controle Integrado de Pragas (CIP). O mofo branco, causado por Sclerotiniasclerotiorum (Lib.), causa danos em culturas de feijão. Este fungo é encontrado no solo e seus sintomas são caracterizados por lesões úmidas cobertas por micélios algodonosos, crescidos a partir do solo e/ou da planta hospedeira. Há relatos de pesquisas buscando agentes potenciais de controle biológico isolados de plantas para controlar fungos fitopatogênicos. Entre estes agentes encontram-se os microrganismos endofíticos, habitantes de espaços intercelulares de tecidos vegetais, muitas vezes responsáveis pela produção de substâncias antimicrobianas. Este trabalho teve por objetivo selecionar linhagens endofíticas isoladas de folhas de confrei (Symphytumofficinale L.), com potencial de antagonismo in vitro contra a linhagem fitopatogênica S. sclerotiorum. Doze linhagens de fungos endofíticos foram utilizadas na técnica de desafio em placa contra um isolado patogênico de S. sclerotiorum. Com o auxílio desta técnica, quatro linhagens com melhor atividade antagonística contra S. sclerotiorum foram selecionadas. Zonas de inibição no crescimento da linhagem patogênica foram consideradas como indicativo de antibiose. Foram efetuadas análises da porcentagem de elongação micelial com e sem antagonismo, mostrando resultados de 46,7% a 50,0% de redução no crescimento micelial do fitopatógeno. Estas análises consistiram de avaliações das medidas do crescimento em mm/dia das linhagens endofíticas/patogênica em testes de antagonismo.

Biological control consists of using one organism to attack another that may cause economic damage to crops. Integrated Pest Management (IPM) is a very common strategy. The white mold produced by Sclerotiniasclerotiorum (Lib.) causes considerable damage to bean crops. This fungus is a soil inhabitant, the symptoms of which are characterized by water-soaked lesions covered by a white cottony fungal growth on the soil surface and/or the host plant. Possible biological control agents taken from plants are being investigated as phytopathogen inhibitors. These are endophytic microorganisms that inhabit the intercellular spaces of vegetal tissues and are often responsible for antimicrobial production. The objective of the present study was to select endophytic fungi isolated from comfrey (Symphytumofficinale L.) leaves with in vitro antagonist potential against the phytopathogenic fungus S. sclerotiorum. Twelve isolates of endophytic fungi and a pathogenic strain of S. sclerotiorum were used in the challenge method. With the aid of this method, four endophytes with the best antagonistic activity against S. sclerotiorum were selected. Pathogen growth inhibition zones were considered indicative of antibiosis. The percentages of pathogenic mycelia growth were measured both with and without the antagonist, resulting in growth reductions of 46.7% to 50.0% for S. sclerotiorum. These analyses were performed by evaluating the endophytic/pathogenic mycelia growth in mm/day over an eight-day period of antagonistic tests. O controle biológico consiste no uso de organismos que atacam outros que causam danos a culturas de plantas. Esta é uma estratégia muito utilizada no Controle Integrado de Pragas (CIP). O mofo branco, causado por Sclerotiniasclerotiorum (Lib.), causa danos em culturas de feijão. Este fungo é encontrado no solo e seus sintomas são caracterizados por lesões úmidas cobertas por micélios algodonosos, crescidos a partir do solo e/ou da planta hospedeira. Há relatos de pesquisas buscando agentes potenciais de controle biológico isolados de plantas para controlar fungos fitopatogênicos. Entre estes agentes encontram-se os microrganismos endofíticos, habitantes de espaços intercelulares de tecidos vegetais, muitas vezes responsáveis pela produção de substâncias antimicrobianas. Este trabalho teve por objetivo selecionar linhagens endofíticas isoladas de folhas de confrei (Symphytumofficinale L.), com potencial de antagonismo in vitro contra a linhagem fitopatogênica S. sclerotiorum. Doze linhagens de fungos endofíticos foram utilizadas na técnica de desafio em placa contra um isolado patogênico de S. sclerotiorum. Com o auxílio desta técnica, quatro linhagens com melhor atividade antagonística contra S. sclerotiorum foram selecionadas. Zonas de inibição no crescimento da linhagem patogênica foram consideradas como indicativo de antibiose. Foram efetuadas análises da porcentagem de elongação micelial com e sem antagonismo, mostrando resultados de 46,7% a 50,0% de redução no crescimento micelial do fitopatógeno. Estas análises consistiram de avaliações das medidas do crescimento em mm/dia das linhagens endofíticas/patogênica em testes de antagonismo.

The aim of this work was to isolate microorganisms from Brazilian soil contaminated with 2,4-D herbicide, and analyze the efficiency for 2,4D degradation, using high-performance liquid chromatography (HPLC). Serratia marcescens and Penicillium sp had never been reported as able to degrade 2,4-D. The isolated strains represent a great potential for bioremediation.O objetivo deste trabalho foi isolar microrganismos de solo brasileiro contaminado com o herbicida 2,4-D, e analisar a eficiência da degradação por cromatografia líquida de alta eficiência (HPLC). Serratia marcescens e Penicillium sp jamais haviam sido relatadas como degradadoras de 2,4-D. As linhagens isoladas representam um grande potencial em biorremediação.

Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max). Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction) primer set and seed soaking (without DNA extraction) for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence) and one naturally infected seed in a 300-seed sample (0.33 % incidence). The PCR-based assay was rapid (< 9 h), did not require DNA extraction and was very sensitive.

In the present study, we have analyzed transformed plants of the citrus rootstock Carrizo citrange constitutively expressing a Δ1-pyrroline-5-carboxylate synthetase mutant gene (p5cs), which encodes the rate-limiting enzyme of the proline biosynthetic pathway. Transgenic plants grew normally in vivo and accumulated high levels of proline in the leaves. In well-watered conditions, the photosynthetic rate of Carrizo citrange transgenic plants was not significantly different from the non-transformed control. The transgenic plants presented superior osmotic adjustment and significantly higher photosynthetic rate than the control plants after 15 days without watering. Under water deficit, the leaf water potential of non-transformed plants was close to zero after 17 days, and the photosynthetic rate was almost null 2 days later. On the other hand, in transgenic plants similar measurements were only observed after 24 days of water stress. The use of citrus rootstocks with increased proline accumulation appears to be a promising approach to maintain the productivity of plants under drought stress condition.

You've heard it several times: the graduate student in Dr. Krebs's lab who made a significant finding with a newly acquired collaborator and published a groundbreaking paper; or Lisa, who started a blog after she met Steve, a program coordinator in science communication, while in circuit training class. Networking creates new opportunities and therefore it's very important to your research and your overall career.

The intensive use of agrochemicals has played an important role in increasing agricultural production. One of the impacts of agrochemical use has been changes in population structure of soil microbiota. The aim of this work was to analyze the adaptive strategies that bacteria use to overcome oxidative stress caused by mesotrione, which inhibits 4-hydroxyphenylpyruvate dioxygenase. We also examined antioxidative stress systems, saturation changes of lipid membranes, and the capacity of bacteria to degrade mesotrione. Escherichia coli DH5-a was chosen as a non-environmental strain, which is already a model bacterium for studying metabolism and adaptation. The results showed that this bacterium was able to tolerate high doses of the herbicide (10× field rate), and completely degraded mesotrione after 3 h of exposure, as determined by a High Performance Liquid Chromatography. Growth rates in the presence of mesotrione were lower than in the control, prior to the period of degradation, showing toxic effects of this herbicide on bacterial cells. Changes in the saturation of the membrane lipids reduced the damage caused by reactive oxygen species and possibly hindered the entry of xenobiotics in the cell, while activating glutathione-S-transferase enzyme in the antioxidant system and in the metabolizing process of the herbicide. Considering that E. coli DH5-α is a non-environmental strain and it had no previous contact with mesotrione, the defense system found in this strain could be considered non-specific. This bacterium system response may be a general adaptation mechanism by which bacterial strains resist to damage from the presence of herbicides in agricultural soils.

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella ) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (“espinheira-santa”) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense . Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Col1 and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

The aim of this work was to isolate microorganisms from Brazilian soil contaminated with 2,4-D herbicide, and analyze the efficiency for 2,4D degradation, using high-performance liquid chromatography (HPLC). Serratia marcescens and Penicillium sp had never been reported as able to degrade 2,4-D. The isolated strains represent a great potential for bioremediation. O objetivo deste trabalho foi isolar microrganismos de solo brasileiro contaminado com o herbicida 2,4-D, e analisar a eficiência da degradação por cromatografia líquida de alta eficiência (HPLC). Serratia marcescens e Penicillium sp jamais haviam sido relatadas como degradadoras de 2,4-D. As linhagens isoladas representam um grande potencial em biorremediação.

Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.

The aim of this work was to study herbicide degradation through selected microorganisms from humus and soil subjected to different plantation systems. The following bacterial species were identified: Klebsiella pneumoniae pneumoniae GC s.B strain 1, Pseudomonas alcaligenes, Enterobacter aerogenes GC s.A and Klebsiella pneumoniae pneumoniae GC s.B strain 2. Growth studies yet suggested the possibility of a very long lag phase. Although, culture with the herbicide presented biofilm formation and there were color changes in the herbicide that could have interfered with the espectrophotometry readings. After 5 days of incubation at 35ºC, the difference in the concentration of herbicide was 14.42% on average and after 10 days, 35.01%.Os herbicidas representam 65% do consumo geral, sendo que o S-Metolachlor é um dos mais utilizados e está trazendo preocupações ambientais. Objetivamos detectar a degradação do S-Metolachlor por microorganismos de solos sob plantio. Foram identificadas as espécies bacterianas: Klebsiella pneumoniae pneumoniae GC s.B linhagem 1, Pseudomonas alcaligenes, Enterobacter aerogenes GC s.A e Klebsiella pneumoniae pneumoniae GC s.B linhagem 2. Resultados da curva de crescimento por espectrofotometria não permitiram definir diferentes fases, levando a pensar em uma fase Lag longa. Frascos de cultura demonstraram a formação de biofilme, provocando mudança na cor do herbicida, interferindo na leitura do crescimento. É possível a existência de fase Log, mas não detectável pelo método. Após 5 dias de incubação a 35ºC, a diferença média de concentração do S-Metolachlor foi de 14.42%, e em 10 dias, 35.01%. Observou-se o aparecimento de um halo em volta das colônias, o que corrobora a hipótese de degradação microbiana do herbicida.

The herbicides ametryn and clomazone are widely used in sugarcane cultivation, and following microbial degradation are considered as soil and water contaminants. The exposure of microorganisms to pesticides can result in oxidative damage due to an increase in the production of reactive oxygen species (ROS). This study investigated the response of the antioxidant systems of two bacterial strains tolerant to the herbicides ametryn and clomazone. Bacteria were isolated from soil with a long history of ametryn and clomazone application. Comparative analyses based on 16S rRNA gene sequences revealed that strain CC07 is phylogenetically related to Pseudomonas aeruginosa and strain 4C07 to P. fulva. The two bacterial strains were grown for 14 h in the presence of separate and combined herbicides. Lipid peroxidation, reduced glutathione content (GSH) and antioxidant enzymes activities were evaluated. The overall results indicated that strain 4C07 formed an efficient mechanism to maintain the cellular redox balance by producing reactive oxygen species (ROS) and subsequently scavenging ROS in the presence of the herbicides. The growth of bacterium strain 4C07 was inhibited in the presence of clomazone alone, or in combination with ametryn, but increased glutathione reductase (GR) and glutathione S-transferase (GST) activities, and a higher GSH concentration were detected. Meanwhile, reduced superoxide dismutase (SOD), catalase (CAT) and GST activities and a lower concentration of GSH were detected in the bacterium strain CC07, which was able to achieve better growth in the presence of the herbicides. The results suggest that the two bacterial strains tolerate the ametryn and clomazone herbicides with distinctly different responses of the antioxidant systems.

Sclerotinia sclerotiorum (Lib.) De Bary é um fungo fitopatogênico filamentoso conhecido como mofo branco. O fungo produz estruturas de resistência chamadas escleródios, que podem permanecer viáveis no solo por anos. O manejo da doença é baseado na rotação de culturas e no uso de fungicidas, os quais contaminam o meio ambiente e selecionam isolados resistentes. Portanto, o desenvolvimento de novos produtos de origem natural para o controle desse fitopatógeno é importante. Nesse contexto, o presente estudo teve como objetivo avaliar o efeito antifúngico de moléculas bioativas produzidas por Pseudomonas aeruginosa cepa LV e Burkholderia metallica cepa RV7S3 sobre a germinação carpogênica de escleródios de Sclerotinia sclerotiorum e seu potencial para o controle da infecção em feijoeiro. A fração com atividade antifúngica produzida pela cepa LV foi denominada F4A, e as frações produzidas pela cepa RV7S3 foram denominadas FD e FRV7. Esta última é uma fração semi-purificada a partir de FD, e apesar de ter efeito antifúngico com baixas concentrações em testes in vitro de ágar difusão, apresenta baixo rendimento, portanto não foi utilizada em testes com gerbox e casa de vegetação. Escleródios produzidos em substrato à base de cenoura foram inoculados em gerbox e tratados com F4A e FD separadamente. Ao final de 55 dias de incubação, foi possível observar inibição crescente da germinação carpogênica dos escleródios, com 100% de inibição nos gerbox tratados com as frações. Folhas destacadas de feijoeiro foram infectadas com S. sclerotiorum e tratadas com as moléculas bioativas. Ambas as frações controlaram o desenvolvimento do fungo, no entanto, F4A a pesar de impedir a infecção fúngica, apresentou fitotoxicidade nas folhas tratadas, portanto os testes em casa de vegetação foram realizados apenas com a fração FD produzida por B. metallica cepa RV7S3. Plantas de feijão foram infectadas com mofo branco e realizado tratamento preventivo+curativo com a aplicação de FD, mantendo 40% das plantas viáveis sem causar fitotoxicidade e impedindo o desenvolvimento fúngico. A fração F4A apresenta potencial para o controle de S. sclerotiorum, no entanto novas estratégias para diminuir a fitotoxicidade devem ser consideradas. O tratamento com os metabólitos secundários produzidos pela cepa RV7S3 apresentaram efeito antifúngico sem causar danos na planta, no entanto estudos futuros precisam ser realizados para otimizar a produção e aumentar o rendimento da fração FRV7, pois esta possivelmente apresentaria maior porcentagem de sobrevivência de plantas em concentrações menores de produto. Sclerotinia sclerotiorum (Lib.) De Bary is a filamentous phytopathogenic fungus known as white mold. The fungus produces resistance structures called sclerotia, which can remain viable in the soil for years. The management of the disease is based on crop rotation and the use of fungicides, which contaminate the environment and select resistant isolates. Therefore, the development of new products of natural origin to control this phytopathogen is important. In this context, the present study had as objective to evaluate the antifungal effect of bioactive molecules produced by Pseudomonas aeruginosa strain LV and Burkholderia metallica strain RV7S3 on the carpogenic germination of Sclerotinia sclerotiorum sclerotia and its potential for controlling infection in bean plants. The fraction with antifungal activity produced by the LV strain was called F4A, and the fractions produced by the RV7S3 strain were called DP and FRV7. The latter is a semi-purified fraction from DP, and despite having an antifungal effect with low concentrations in vitro diffusion agar tests, it has low yield, therefore it was not used in tests with gerbox and greenhouse. Sclerotia produced on carrot-based substrate were inoculated in gerbox and treated with F4A and DP separately. At the end of the 55 days of incubation, it was possible to observe increasing inhibition of carpogenic germination of sclerotia, with 100% inhibition in the gerbox treated with the fractions. Detached bean plants leaves were infected with S. sclerotiorum and treated with bioactive molecules. Both fractions controlled the development of the fungus, however, F4A despite preventing fungal infection, showed phytotoxicity in the treated leaves, so the tests in the greenhouse were performed only with the DP fraction produced by B. metallica strain RV7S3. Bean plants were infected with white mold and performed preventive+curative treatment with DP application, maintaining 40% of viable plants without causing phytotoxicity and preventing fungal development. The F4A fraction has the potential to control of S. sclerotiorum, however new strategies to decrease phytotoxicity must be considered. The treatment with secondary metabolites produced by the RV7S3 strain showed antifungal effect without causing damage to the plant, however future studies need to be carried out to optimize the production and increase the yield of the FRV7 fraction, as this would possibly present a better percentage of plant survival in lower concentrations of product.

Pseudomonas aeruginosa é amplamente dispersa na natureza, devido principalmente à sua alta capacidade adaptativa. Dentre as diversas estratégias de adaptação está a produção de metabólitos bioativos que podem inibir o crescimento ou metabolismo de outros microrganismos. P. aeruginosa cepa LV produz diversos compostos bioativos, entre eles um composto organometálico, identificado como Fluopsina C, produzido somente na presença de cobre, que apresenta alto potencial biotecnológico. Entretanto, poucos são os relatos sobre esta molécula, não havendo informações relacionadas à sua biossíntese e rota metabólica. O objetivo deste trabalho foi realizar o sequenciamento completo do genoma de P. aeruginosa cepa LV e realizar a análise da expressão temporal dos genes diferencialmente expressos pela presença/ausência de cobre, afim de melhor compreender a biossíntese da Fluopsina C. Para isso, a cepa foi cultivada em ágar nutriente overnight e após a extração do DNA total do microrganismo foi realizado o sequenciamento pela plataforma Illumina MiSeq e o genoma foi montado pela estratégia de novo, filtrado, trimado e anotado. O genoma de P. aeruginosa cepa LV é constituído por 6.468.334 pb e, possue 14 grupos de genes putativos responsáveis pela biossíntese de metabólitos secundários, 7 genes de resistência e 3 arranjos CRISPRs (Clustered Regularly Interspaced Regularly Short Palindromic Repeats). A análise da expressão temporal foi realizada por PCR em tempo real e com pontos de análise em 24 horas, 4 e 7 dias. Foram estudados nove genes hiperexpressos na presença de cobre, divididos em três classes: transportadores de metais, reguladores de expressão e biossíntese de fenazina e proteínas hipotéticas. Os resultados obtidos validam as hipóteses geradas em trabalhos anteriores com relação a eliminação do cobre através de efluxo e transportadores ATPases. Além disso, sugerimos uma nova hipótese com relação a via de biossíntese da Fluopsina C, relacionando a produção de compostos fenazínicos ao percursor da molécula, a tioformina. Pseudomonas aeruginosa is widespread, due to its high adaptive capacity. Among the several adaptation strategies, the production of bioactive metabolites can inhibit microbial growth or metabolic pathways. P. aeruginosa LV strain produces several bioactive compounds, including an organometallic compound, Fluopsin C, that is produced only in the presence of copper and shows high biotechnological potential. However, there are few reports in the literature about this molecule, including its biosynthesis and metabolic route. The objective of this study was to sequence the complete genome of P. aeruginosa strain LV and to perform the analysis of the temporal expression of genes differentially expressed by the presence/ absence of copper, in order to understand the Fluopsin C biosynthesis. For this, the strain was grown on nutrient agar overnight and after extracting the total DNA the sequencing was performed using Illumina MiSeq platform, and then the genome was assembled using the De Novo strategy, filtered, trimmed, and annotated. The final assembly of the genome was 6,468,334 bp, where 14 groups of putative genes responsible for the biosynthesis of secondary metabolites, 7 antimicrobial resistance genes, and 3 CRISPRs (Clustered Regularly Interspaced Regularly Short Palindromic Repeats) arrangements were located. Temporal expression analysis was performed by real-time PCR and with analysis points in 24 hours, 4, and 7 days. Nine overexpressed genes were studied in the presence of copper, divided into 3 classes: metal transporters, gene expression regulators and, phenazine biosynthesis and hypothetical proteins. The results obtained validate the hypotheses generated in previous studies regarding the elimination of copper through efflux and ATPase transporters. Also, we suggest a new hypothesis regarding the biosynthesis pathway of Fluopsin C, relating the production of phenazine compounds to the precursor of the molecule, thioformin.

A contaminação ambiental por pesticidas é uma preocupação cada vez mais presente, como indicam estudos que detectam frequentemente estes compostos em água e solo, e visto a importância destes, faz-se necessário a compreensão do seu impacto em organismos não alvo. Dessa forma, o estudo da resposta adaptativa bacteriana a herbicidas vem de encontro com a necessidade descrita, além de fornecer novos dados a cerca da regulação metabólica na possível biotransformação destes compostos. Neste estudo, o sistema antioxidante foi alvo de análises para a compreensão de sua participação neste processo, além de evidenciar a regulação diferencial de isoenzimas em bactéria exposta à herbicida. Para tanto, inicialmente um estudo de biodegradação apontou que o isolado bacteriano Enterobacter asburiae SD1 é capaz de degradar s-metolachlor em cerca de 30% com 13 h de cultivo em meio mineral, na presença de 1,25 mM do herbicida. Porém, a biodegradação também ocorreu em meio de cultura com fonte de carbono alternativa ao s-metolachlor, o que juntamente com o crescimento diferencial entre os meios, sugeriu uma resposta fisiológica adaptativa à diferentes condições nas quais o herbicida estava presente. Conhecido em estudos de resposta global a estresse, o sistema antioxidante foi avaliado na presença do herbicida e em diferentes consituições de meio de cultura, o que culminou em vias preferenciais ativadas conforme as características dos meios. Na presença de somente s-metolachlor como fonte de carbono (meio mineral), a enzima glutation S-transferase foi ativada, mostrando prioridade no reconhecimento e metabolização do s-metolachlor. Já na presença de outras fontes de carbono, além do herbicida, as vias de combate ao estresse oxidativo, representadas pelas enzimas catalase (CAT), superóxido dismutase (SOD) e glutationa redutase (GR), foram ativadas primeiramente. Além disso, foi possível também avaliar a ativação diferencial das isoenzimas de CAT e SOD frente ao s-metolachlor, sendo que a isoenzima Mn-SOD merece destaque pela importância na resposta adaptativa, visto que a deleção do gene sodA (Mn- SOD), causou danos a membrana e desequilíbrio no trabalho cooperativo enzimático no combate às ERO\'s (espécies reativas de oxigênio). Finalmente, a integração dos diferentes níveis de um metabolismo na resposta do sistema antioxidante bacteriano foi mostrado através do estudo com a via de controle traducional formada pela proteína EF-P (elongation factor P). Esta proteína atua na fidelidade do processo de síntese de proteína, e a deleção do gene efp que a codifica, bem como outras proteínas envolvidas na ativação desta via, resultou no desequilíbrio celular, gerando estresse oxidativo, potencializado pela presença do herbicida paraquat. Este trabalho reúne resultados bastante relevantes para a compreensão do papel do sistema antioxidante bacteriano, em condições de estresse gerado pela presença de herbicidas, e oferece dados importantes referentes a integração e regulação de vias bioquímicas na ativação do sistema antioxidante, bem como para uma eficiente resposta adaptativa deste sistema. The environmental contamination by pesticides is an increasing concern, as frequently showed in studies with detection of these compounds in water and soil. In fact, the understanding of pesticide effect on non-target organisms is necessary given the importance of pesticide and its sustentable use. The study of bacterial adaptive response to pesticide can show how the metabolic regulation happens, as well as provide new data about biotransformation of these xenobiotics. For this propose, firstly the biodegradation analyse showed the Enterobacter asburiae SD1 strain has capacity to degrade the s-metolachlor herbicide in around 30% of your initial concentration (1,25 mM), in 13 h of cultivation in mineral medium. However, the degradation was also observed on the presence of alternative carbon source, what suggests, together the differential growth, the bacteria can show an adaptive response to different conditions represented by the media constitution. The antioxidant system, a global sensor to stress, was evaluated in E. asburiae SD1 cultivated in different media, to see what is the effect of s-metolachlor herbicide in the antioxidant response. It was possible to observe preferential pathways activated according to medium characteristics, such as the activation of glutathione Stransferase in the presence of only s-metolachlor as carbon source. This result was explained by the bacterial need to recognize and metabolize a new molecule. On the other hand, in the presence of alternative carbon source, in addition to herbicide, the pathways to combat oxidative stress, as those constituted by catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), were firstly activated. Besides this, the differential activation was observed among the isoenzymes of CAT and SOD, wich special attention to Mn-SOD, which one had an important role in the adptive responde to herbicide. This affirmation was justified when de delection of sodA gene (Mn-SOD) induced membrane damage and disruption in the cooperative work of antioxidant enzymes in bacteria exposed to s-metolachlor. Finally, the integration of different levels of metabolism in the antioxidant response was showed through study with a pathway envolved in the translational control which has the elongation factor-P (EFP) as one important protein. This enzyme acts in the fidelity during the protein synthesis, and its absence, caused by efp gene delection, as well others two protein important to activate the EFP, induced the oxidative stress, increased by the paraquat herbicide. The present work collects relevant results for understanding of the role of bacterial antioxidant system in the stress condition induced by herbicide presence, and also offers important data about integration and regulation of biochemical pathway in the antioxidant system activation for an efficient adaptative response.

Os herbicidas ametrina e clomazone são amplamente utilizados na cultura de cana-deaçúcar, apresentam degradação essencialmente microbiana e são considerados contaminantes de solos e águas. A exposição dos microrganismos a estes xenobióticos pode resultar em dano oxidativo devido ao aumento na produção de espécies reativas de oxigênio (EROs). Este trabalho teve como objetivo analisar respostas bioquímicas e fisiológicas de dois isolados bacterianos tolerantes aos herbicidas ametrina e clomazone. As bactérias foram isoladas de solos agrícolas e a partir de estudos fisiológicos, crescimento e formação de halo, foram selecionadas duas que apresentaram maior tolerância aos herbicidas. Esses microrganismos foram cultivados em meio de cultura ágar nutriente, a 30°C, por 14 horas na presença dos herbicidas ametrina (25 mM), clomazone (9 mM), e em meio composto pelos dois herbicidas: ametrina (20 mM) + clomazone (20 mM). Essas concentrações representam as dosagens utilizadas para o cultivo de cana-deaçúcar para a aplicação pré e pós-emergência. Por meio da análise filogenética baseada no gene 16S ribossômico, o isolado bacteriano CC07 mostrou-se próximo a Pseudomonas aeruginosa, enquanto que o isolado 4C07 ficou próximo à Pseudomonas fulva. A peroxidação lipídica foi observada somente para o isolado CC07 na presença dos herbicidas em mistura. O perfil protéico foi avaliado em SDS-PAGE, o qual revelou a indução de uma nova proteína de aproximadamente 60 KDa para o isolado 4C07 na presença dos herbicidas, porém, não foram observadas diferenças significativas para o isolado CC07. A enzima superóxido dismutase (SOD) apresentou aumento significativo da atividade para ambos isolados quando expostos aos herbicidas, entretanto, diferenças na atividade da enzima catalase (CAT) não foram observadas. Para o isolado 4C07 na presença dos herbicidas foi observado aumento significativo no conteúdo de glutationa reduzida (GSH), o qual foi acompanhado pela indução de uma nova isoforma de GR (I), que respondeu especificamente ao clomazone e os herbicidas em mistura. A glutationa-S-transferase (GST) também apresentou acréscimos de atividade quando o isolado 4C07 foi exposto aos herbicidas. Porém, para o isolado CC07 as enzimas GR, GST, assim, como a GSH não apresentaram respostas significativas a presença dos herbicidas. Assim, foi possível observar que o isolado 4C07 possui um sistema antioxidante mais eficaz, quando comparado com o isolado CC07. Além disso, os resultados sugerem que as enzimas SOD, GR, GST e o composto GSH podem estar relacionados com o mecanismo de tolerância do isolado 4C07 aos herbicidas ametrina e clomazone, o que pode demonstrar uma provável adaptação aos ambientes estressantes. The herbicides ametrina and clomazone are widely used in the sugar cane cultivation, showing essential microbial degradation, besides being considered contaminants of soil and water. The exposure of microorganisms to xenobiotics can result in oxidative damage due to increased production of reactive oxygen species (ROS). This study aimed to analyze biochemical and physiological responses of two bacterial strains tolerant to the herbicides ametrina and clomazone. The bacteria strains were isolated from agricultural soils and according to physiological studies, growth and halo formation, two of them with the most herbicide tolerance were selected. These microorganisms were grown in nutrient agar plates at 30 ° C for 14 hours in the presence of herbicides ametrina (25 mM), clomazone (9 mM), and in a medium composed by two herbicides: ametrina (20 mM) + clomazone (20 mM). These concentrations represent the concentrations used for growing sugar cane for pre and post-emergence. Through the phylogenetic analysis based on 16S ribosomal gene, the CC07 strain was close to Pseudomonas aeruginosa, while the 4C07 strain was close to Pseudomonas fulva. According to the results obtained, lipid peroxidation was only observed for CC07 strain in a medium composed by two herbicides. The protein profile was evaluated by SDS-PAGE, which revealed the induction of a new protein of approximately 60 kDa for 4C07 strain in the presence of the two herbicides, differently to that observed for CC07 strain. The activity of the enzyme superoxide dismutase (SOD) increased significantly for both strains when exposed to the herbicides, however, no differences were observed for catalase (CAT) activity. The 4C07 strain showed increase in content of reduced glutathione (GSH) in the presence of the herbicides, which was accompanied by the induction of a new isoform of GR (I). Similarly, the 4C07 strain exhibited an increase in Glutathione-S-transferase (GST) activity to herbicides exposure. However, for the CC07 strain, the GR and GST enzymes, as well as the GSH content did not exhibit differences. Thus, we observed that the 4C07 strain may have an antioxidant system more effective when compared to the CC07 strain. Moreover, the results suggest that the enzymes SOD, GR, GST and GSH content may be related to the mechanism of tolerance of 4C07 strain to ametrina and clomazone herbicides, showing the tendency to deal better and adapt to stressful environments.