Miguel O. P. Navarro

Bioactive Organocopper Compound from Pseudomonas aeruginosa Inhibits the Growth of Xanthomonas citri subsp. citri

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.

The Effect of Phenazine-1-Carboxylic Acid on Mycelial Growth of Botrytis cinerea Produced by Pseudomonas aeruginosa LV Strain

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.

Control of bacterial stem rot on tomato by extracellular bioactive compounds produced by Pseudomonas aeruginosa LV strain

Abstract This study evaluated the antibiotic activity and induction of resistance in plants by compounds produced by Pseudomonas aeruginosa LV strain on the control of bacterial stem rot in tomato. Compounds were extracted from the cell-free supernatant of a bacterial culture and purified. The F4A fraction was composed of two major compounds, an antibiotic and a phenazine (PCN). The first experiment evaluated the antibiotic activity of F4A, and the second one compared the ability of F4A and PCN to elicit systemic acquired resistance (SAR). In both experiments, plants were infected with Pectobacterium carotovorum subsp. carotovorum (Pcc). The minimum inhibitory concentration of F4A was 7.81 μg mL−1, and PCN did not inhibit bacterial growth. The results suggest that F4A has antibiotic activity. Scanning electron microscopy revealed changes in bacterial cells after 3 h treatment with F4A. F4A and PCN decreased symptoms of stem rot and increased fruit production. Plant response was estimated by determination of peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase activity. Plants treated with PCN or F4A showed greater enzyme activity than plants that were not treated or treated with Bion®, suggesting that PCN increased SAR. The compounds showed the potential to control Pcc in vitro and in vivo and to induce plant response.

Low-molecular-weight metabolites produced by Pseudomonas aeruginosa as an alternative to control Huanglongbing in Citrus sinensis cv. Valencia

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.

Disease-Induced Resistance and Plant Immunization Using Microbes

The induction of resistance in plants presents as an alternative to be explored in several species. This process involves the activation of defense mechanisms, which are inactive or latent in the plant and do not require alterations in your genome. This activation can be effected by biotic and abiotic agents known as resistance inducers. The use of resistance inducers leads to activation of the systemic resistance, which leads to a marked reduction in symptoms of the disease after subsequent infections, including different species of pathogens. This chapter gathers information about diverse compounds of biological origin that can act as resistance inductors, as well as an interaction between plants and rhizosphere microorganisms that may result in the activation of this resistance system against pathogens.

Strategies for Biological Control and Antagonisms

Microorganisms play an important niche in the control of soil populations producing a variety of bioactive compounds as an ecological strategy for competition for space and nutrients. Thus, the bioprospecting of microorganisms as potential antagonists for pathogen biocontrol, or obtaining their bioactive metabolites, is one of the alternatives currently studied for the control of diseases, especially in species of agronomic importance. In this chapter, we reviewed several microorganisms and how, in general, the products of their metabolism are obtained to be used in the control of pathogens.

New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis

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.

Utilização de compostos naturais extraídos de Pseudomonas aeruginosa no incremento da produtividade do milho (Zea mays L.) e da soja (Glycine max L.)

Introducao: O milho (Zea mays L.) e a soja (Glycine max L.) sao duas das mais importantes cultivares na agricultura brasileira, e demandam grandes quantidades de agroquimicos, provocando tanto problemas ambientais quanto problemas a saude. O objetivo deste trabalho foi avaliar compostos naturais, extraidos de Pseudomonas aeruginosa cepa LV com potencial para controle de doencas e consequente incremento da produtividade, como alternativa ao uso de fungicidas comerciais. Metodo: A cepa LV foi isolada a partir de lesao velha de cancro citrico em fruto de laranja caido, no municipio de Astorga – PR (Rampazo, 2004), e e mantida no Laboratorio de Ecologia Microbiana. A producao dos bioativos foi realizada a partir de cultivo da cepa LV em caldo nutriente suplementado com CuCl2. A cultura foi incubada a 28 °C, 10 dias. Apos o cultivo, as celulas foram separadas por centrifugacao e o sobrenadante foi concentrado a 10% do volume inicial e submetido a extracao com diclorometano, obtendo, assim, o composto bioativo FD. O experimento foi realizado na area experimental da fazenda escola da Universidade Estadual de Londrina – UEL, no periodo entre dez/2014 e mai/2015 sob delineamento em blocos casualizados, onde foram testados 4 diferentes tratamentos distribuidos em 4 blocos, tanto para milho quanto para a soja, sendo eles: testemunha absoluta (T1), fungicida comercial Unizeb Gold, Sphere Max e Opera 1,5Kg/ha (T2), FD 100μg/mL (T3), FD 10μg/mL(T4). Ao final do experimento as plantas foram avaliadas quanto a producao total de graos. Resultados: A cultura do milho nao apresentou diferenca estatistica entre os tratamentos, ja para a cultura da soja o T2 apresentou a maior taxa de produtividade, seguida do T4, T3 e finalmente o T1. Conclusoes: Os dados obtidos indicam que a concentracao da FD pode estar relacionada ao indice de produtividade, e sugerem que leguminosas podem responder melhor ao composto natural em relacao a nao-leguminosas. Agencia de Fomento: Capes

PLANT GROWTH-PROMOTING MICROBIAL INOCULANT FOR Schizolobium parahyba pv. parahyba

Schizolobium parahyba pv. amazonicum (Huber ex Ducke) Barneby (parica) occurs naturally in the Amazon and is significant commercial importance due to its rapid growth and excellent performance on cropping systems. The aim of this paper was to evaluate a microbial inoculants such as arbuscular mycorrhiza fungi (AMF) and Rhizobium sp. that promote plant growth. The inocula was 10 g of root colonized and spores of Glomus clarum and/or 1 mL of cell suspension (107 CFU/mL) of Rhizobium sp. and/or 100 g of chemical fertilizer NPK 20-05-20 per planting hole. The experimental design was complete randomized blocks with five replications and eight treatments (n = 800). Plant height, stem diameter and plant survival were measured. The results were tested for normality and homogeneity of variances and analyzed by ANOVA and Tukey test (p < 0.05). Rhizobium sp and AM fungi showed no effect on plant growth. Environmental factors probably influenced the effectiveness of symbiosis of both microorganisms and plant growth. The chemical fertilizer increased S. parahyba growth. During the first 120 days plants suffered with drought and frost, and at 180 days plants inoculated with microorganism plus chemical fertilizer showed higher survival when compared with control. The results showed that the microbial inoculants used showed an important role on plant survival after high stress conditions, but not in plant growth. Also was concluded that the planting time should be between November to December to avoid the presence of young plants during winter time that is dry and cold.