One of the most important postharvest plant pathogens that affect strawberries, grapes and tomatoes is Botrytis cinerea, known as gray mold. The fungus remains in latent form until spore germination conditions are good, making infection control difficult, causing great losses in the whole production chain. This study aimed to purify and identify phenazine-1-carboxylic acid (PCA) produced by the Pseudomonas aeruginosa LV strain and to determine its antifungal activity against B. cinerea. The compounds produced were extracted with dichloromethane and passed through a chromatographic process. The purity level of PCA was determined by reversed-phase high-performance liquid chromatography semi-preparative. The structure of PCA was confirmed by nuclear magnetic resonance and electrospray ionization mass spectrometry. Antifungal activity was determined by the dry paper disk and minimum inhibitory concentration (MIC) methods and identified by scanning electron microscopy and confocal microscopy. The results showed that PCA inhibited mycelial growth, where MIC was 25 μg mL-1. Microscopic analysis revealed a reduction in exopolysaccharide (EPS) formation, showing distorted and damaged hyphae of B. cinerea. The results suggested that PCA has a high potential in the control of B. cinerea and inhibition of EPS (important virulence factor). This natural compound is a potential alternative to postharvest control of gray mold disease.

Citrus canker is a very destructive disease of citrus species. The challenge is to find new compounds that show strong antibiotic activity and low toxicity to plants and the environment. The objectives of the present study were (1) to extract, purify and evaluate the secondary metabolites with antibiotic activity produced by Pseudomonas aeruginosa LV strain in vitro against Xanthomonas citri subsp. citri (strain 306), (2) to determine the potential of semi-purified secondary metabolites in foliar application to control citrus canker under greenhouse conditions, and (3) to identify antibiotic activity in orange leaf mesophyll infected with strain 306, by electron microscopy. Two pure bioactive compounds were isolated, an organocopper antibiotic compound (OAC) and phenazine-1-carboxamide. Phenazine-1-carboxamide did not show any antibiotic activity under the experimental conditions used in this study. The OAC showed a high level of antibiotic activity with a minimum inhibitory concentration of 0.12 μg mL-1. In greenhouse tests for control of citrus canker in orange trees, the semi-purified fraction F3d reduced lesion formation by about 97%. The concentration used was 500 times lower than that for the recommended commercial copper-based product. Electron microscopy showed that F3d altered the exopolysaccharide matrix and caused cell lysis of the pathogen inside the citrus canker lesions. These results suggest that secondary metabolites produced by inducing P. aeruginosa LV strain have a high potential to be used as a bioproduct to control citrus canker.

This study investigated the protective effects of secondary bacterial metabolites, produced by Pseudomonas sp. (bacterium strain LN), on citrus canker disease caused by Xanthomonas axonopodis pv. citri (Xac 306). The LN bacteria strain was cultured in liquid medium and the supernatant free-cells was treated with methanol (AMF) and ethyl acetate (AEF), respectively, and then the extract was concentrated, filtrated, lyophilized and fractionated by vacuum liquid chromatography (VLC). After VLC, eight fractions were obtained. All fractions' activity against Xac 306 by agar well diffusion assay and minimum inhibitory concentration but in different concentrations were tested. Cytotoxicity effects were observed in all fractions in 50 µg•mL −1 concentration. The comet assay demonstrated that the fractions EAF, VLC2 and VLC3 presented no genotoxic effects at tested concentrations. In plants only VLC3 showed significant results (p < 0.05), reducing the incidence of citrus canker lesions.

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The increasing emergence of multidrug-resistant (MDR) organisms in hospital infections is causing a global public health crisis. The development of drugs with effective antibiotic action against such agents is of the highest priority. In the present study, the action of Fluopsin C against MDR clinical isolates was evaluated under in vitro and in vivo conditions. Fluopsin C was produced in cell suspension culture of Pseudomonas aeruginosa LV strain, purified by liquid adsorption chromatography and identified by mass spectrometric analysis. Bioactivity, bacterial resistance development risk against clinically important pathogenic strains and toxicity in mammalian cell were initially determined by in vitro models. In vivo toxicity was evaluated in Tenebrio molitor larvae and mice. The therapeutic efficacy of intravenous Fluopsin C administration was evaluated in a murine model of Klebsiella pneumoniae (KPC) acute sepsis, using six different treatments. The in vitro results indicated MIC and MBC below 2 μg/mL and low bacterial resistance development frequency. Electron microscopy showed that Fluopsin C may have altered the exopolysaccharide matrix and caused disruption of the cell wall of MDR bacteria. Best therapeutic results were achieved in mice treated with a single dose of 2 mg/kg and in mice treated with two doses of 1 mg/kg, 8 h apart. Furthermore, acute and chronic histopathological studies demonstrated absent nephrotoxicity and moderate hepatotoxicity. The results demonstrated the efficacy of Fluopsin C against MDR organisms in in vitro and in vivo models, and hence it can be a novel therapeutic agent for the control of severe MDR infections.

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The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified 12 upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and we suggesting that may involve in the biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide and an efficient management strategy to control it has not yet been established. The potential of pseudomonads to suppress plant pathogens is well known and the secondary metabolites they produce represent new alternatives of compounds to control plant diseases. The main challenge is to find new compounds that show strong antibiotic activity, low toxicity to plants and little or no harm to the environment. The objectives of the present study were to determine the potential of the F4A fraction from Pseudomonas aeruginosa to control HLB and to induce systemic resistance. Low molecular weight compounds with antimicrobial activity were purified with organic solvent, thin layer chromatography (TLC) and normal and reverse phase chromatography. Compounds present in the F4A fraction were mainly obtained by thin-layer chromatography (TLC) and Preparative-High Performance Liquid Chromatography (HPLC-prep). To assess their biological activities, conventional and quantitative polymerase chain reaction were usxed. The F4A was sprayed on citrus trees infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus' under greenhouse conditions. The bacterial titers were reduced and defense genes were induced in leaves of trees treated with F4A, as assed by PCR analysis. The results showed that F4A (pseudomonads secondary metabolites) may provide a useful tool for the management of HLB.