Comparación de dos métódos de preparación de tejido para estudiar la anatomía interna del delfácido Tagosodes orizicolus con microscopia de luz y electrónica

Two methods of embedding, sectioning and staining were developed to study the intemal anatomy of the delphacid planthopper Tagosodes orizicolus, one of the most important plagues of rice in Latín America and the only vector of the hoja blanca tenuiVirus (RHBV), using both light and electron rnicroscopy. The paraffinehematoxiline- eosine Y Iliethod allows tite color identification of tissues, for example purple for fat tissue, pink for muscles, yellow- brown for exocutícle, while the resin-toluidíne-blue method preserves hetter the ultrastructure but do not permit colór identification. The informatíon obtained by these procedures is complementary and the material can also be used for in situ studies by irnmunomicroscopy, to assess the changes in cell ultrastructure and the localizatíon and replication of the RHBV during its infection cycle in the insect vector.Two methods of embedding, sectioning and staining were developed to study the intemal anatomy of the delphacid planthopper Tagosodes orizicolus, one of the most important plagues of rice in Latín America and the only vector of the hoja blanca tenuiVirus (RHBV), using both light and electron rnicroscopy. The paraffinehematoxiline- eosine Y Iliethod allows tite color identification of tissues, for example purple for fat tissue, pink for muscles, yellow- brown for exocutícle, while the resin-toluidíne-blue method preserves hetter the ultrastructure but do not permit colór identification. The informatíon obtained by these procedures is complementary and the material can also be used for in situ studies by irnmunomicroscopy, to assess the changes in cell ultrastructure and the localizatíon and replication of the RHBV during its infection cycle in the insect vector.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Peptaibol Production and Characterization from Trichoderma asperellum and Their Action as Biofungicide

Peptaibols (Paib), are a class of biologically active peptides isolated from soil, fungi and molds, which have interesting properties as antimicrobial agents. Paib production was optimized in flasks by adding sucrose as a carbon source, 2-aminoisobutyric acid (Aib) as an additive amino acid, and F. oxysporum cell debris as an elicitor. Paib were purified, sequenced and identified by High-performance liquid chromatography (HPLC)coupled to mass spectrometry. Afterward, a Paib extract was obtained from the optimized fermentations. The biological activity of these extracts was evaluated using in vitro and in vivo methods. The extract inhibited the growth of specific plant pathogens, and it showed inhibition rates similar to those from commercially available fungicides. Growth inhibition rates were 92.2, 74.2, 58.4 and 36.2% against Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria alternata and Fusarium oxysporum, respectively. Furthermore, the antifungal activity was tested in tomatoes inoculated with A. alternata, the incidence of the disease in tomatoes treated with the extract was 0%, while the untreated fruit showed a 92.5% incidence of infection Scanning electron microscopy images showed structural differences between the fungi treated with or without Paib. The most visual alterations were sunk and shriveled morphology in spores, while the hyphae appeared to be fractured, rough and dehydrated

Identificación del virus del mosaico del maíz, un rhabdovirus, en Costa Rica

Datos y artículo incluido por Lisela Moreira Carmona, responsable de depósitos de publicaciones del área de Patógenos y Plagas del CIBCMMaize mosaic virus (MMV), a rhabdovirus, was identified associated to maize field plants, showing stunting and continuous chlorotic stripes uniformely distributed over the leaf blade.The virus was detected in field samples by agar-gel immunodifussion. Enveloped, bacilliform virus particles were observed by electron microscopy in thin sections of naturally infected leaf tissue.El rhabdovirus conocido como virus del mosaico del maíz (MMV) fue identificado asociado a plantas de maíz que presentaban en el campo, enanismo y bandas cloróticas continuas, uniformemente distribuidas en la lámina foliar. En corte ultrafinos de tejido foliar en estas plantas se observaron por microscopia electrónica de partículas virales baciliformes con envoltura, de diferentes tamaños dependiendo de su grado de desarrollo. Por el método serológico de inmunodifusión doble en agar, se identificó este virus como el virus del mosaico del maíz.Universidad de Costa Rica/[801-94-905]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Detection of Potyvirus-like Particles Associated with Oil Palms (Elaeis guineensis) in Ecuador

Copyright 1996 The American Phytopathological Society.Oil palm (Elaeis guineensis Jacq.) seedlings showing a conspicuous mottling, composed of alternating dark green and chlorotic areas in the form of discontinuous streaks or irregular ring patterns, were observed in the occidental region of Ecuador. Leaves from normal and symptomatic plants were analyzed by transmission electron microscopy. The leaf-dip preparations and partially purified preparations from symptomatic plants consistently contained filamentous, flexuous, rod-shaped, viral particles of approximately 682 x 13 nm. The size and morphology of the particles resemble those previously reported for palm mosaic virus, a virus that is tentatively placed in the potyviridae (1). Cylindrical inclusion (CI) bodies containing laminated aggregates, nuclear fibrillar inclusion bodies, viral particles scattered in the cytoplasm, and bundles of particles attached to CI plates and to the tonoplast were only observed in thin sections of symptomatic leaf tissue. CI bodies were similar to those reported for palm mosaic virus (1). The size and morphology of the particles and the type of the inclusion bodies observed are similar to those of potyviruses. Therefore, we suggest that the symptoms observed in oil palm are caused by a potyvirus, which is probably palm mosaic virus. This is the first report of a potyvirus infecting Elaeis guineensis in Ecuador.Universidad de Costa Rica/[801-94-905]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Clostridium perfringens Phospholipase C, an Archetypal Bacterial Virulence Factor, induces the Formation of Extracellular Traps by Human Neutrophils

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins, released to the extracellular space to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and is involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen that produces many exotoxins associated with various animal and human diseases, including the necrotizing soft tissue infection called gas gangrene. This work demonstrates that the C. perfringens toxinotype A secretome induces NETs formation (NETosis) in human neutrophils. Antibodies against the C. perfringens phospholipase C (CpPLC) completely abrogate the NETosis-inducing activity of that secretome, and the recombinant CpPLC induces NETs formation in a dose-response manner. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that help other pathogens evade NETs. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/ extracellular signal-regulated kinase (MEK/ERK) pathways, and the production of reactive oxygen species (ROS) by the xanthine oxidase (XO) and the metabolism of arachidonic acid. CpPLC was the first bacterial toxin found to be enzymatically active and is the major virulence factor in the pathogenesis of gas gangrene. This toxin drives the formation of neutrophil/platelet aggregates within the vasculature of the infected tissues, which leads to the circulation's halt and extends the anaerobic environment for C. perfringens growth. It is suggested that this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis, and then escapes and spreads to deeper tissues. Understanding the role of NETs in the thrombotic events occurring in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Differential volatile organic compound expression in the interaction of Daldinia eschscholtzii and Mycena citricolor

Fungi exhibit a wide range of ecological guilds, but those that live within the inner tissues of plants (a.k.a. endophytes) are particularly relevant due to the benefits they sometimes provide to their hosts, such as herbivory deterrence, disease protection, and growth promotion. Recently, endophytes have gained interest as potential biocontrol agents against crop pathogens, for example, coffee plants (Coffea arabica). Published results from research done in our laboratory showed that endophytic fungi isolated from wild Rubiaceae plants were effective in reducing the effects of the American Leaf Spot of Coffee (Mycena citricolor). One of these isolates (GU11N) from the plant Randia grandifolia was identified as Daldinia eschscholtzii (Xylariales). Its antagonism mechanisms, effects, and chemistry against M. citricolor were investigated through the analysis of its volatile profile alone and in the presence of the pathogen in contactless and dual culture assays. The experimental design involved direct sampling of agar plugs in vials for headspace (HS) and headspacesolid- phase-microextraction (HS-SPME) Gas Chromatography Mass Spectrometry (GC-MS) analysis. Additionally, we used UHPLC-HRMS/MS to identify non-volatile compounds from organic extracts of the mycelia involved in the interaction. Results showed that more volatile compounds were identified using HS-SPME (39 components) than with the HS technique (13 components), sharing only 12 compounds. Statistical tests suggest that D. eschscholtzii inhibited the growth of M. citricolor through the release of VOCs containing a combination of 1,8- dimethoxynapththalene and terpene compounds affecting the M. citricolor pseudopilei. The damaging effects of 1,8-dimethoxynaphthalene were corroborated in an in vitro test against M. citricolor pseudopilei; SEM photographs confirmed the structural damage. After analyzing the UHPLC-HRMS/MS data, a predominance of fatty acid derivatives was found among the putatively identified compounds, however, a considerable proportion of features (37.3%) remained unannotated. In conclusion, our study suggests that D. eschscholtzii has a potential as a biocontrol agent against M. citricolor and that 1,8-dimethoxynaphthalene contributes to the observed damage to the pathogen’s reproductive structures.Universidad de Costa Rica/[809-B7-176]/UCR/Costa RicaUniversidad de Costa Rica/[809-C1-604]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de BiologíaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Optimization of the biosynthesis of silver nanoparticles using bacterial extracts and their antimicrobial potential

In the present study, silver nanoparticles (AgNPs) were biosynthesized using the supernatant and the intracellular extract of Cupriavidus necator, Bacillus megaterium, and Bacillus subtilis. The characterization of the AgNPs was carried out using UV–Vis spectroscopy, FTIR, DLS and TEM. Resazurin microtiter-plate assay was used to determine the antimicrobial action of AgNPs against Escherichia coli. UV–Visible spectra showed peaks between 414 and 460 nm. TEM analysis revealed that the synthesized AgNPs showed mostly spherical shapes. DLS results determined sizes from 20.8 to 118.4 nm. The highest antimicrobial activity was obtained with the AgNPs synthesized with supernatant rather than those using the intracellular extract. Therefore, it was determined that the bacterial species, temperature, pH, and type of extract (supernatant or intracellular) influence the biosynthesis. This synthesis thus offers a simple, environmentally friendly, and low-cost method for the production of AgNPs, which can be used as antibacterial agents

Differential Volatile Organic Compound Expression in the Interaction of Daldinia eschscholtzii and Mycena citricolor

Fungi exhibit a wide range of ecological guilds, but those that live within the inner tissues of plants (also known as endophytes) are particularly relevant due to the benefits they sometimes provide to their hosts, such as herbivory deterrence, disease protection, and growth promotion. Recently, endophytes have gained interest as potential biocontrol agents against crop pathogens, for example, coffee plants (Coffea arabica). Published results from research performed in our laboratory showed that endophytic fungi isolated from wild Rubiaceae plants were effective in reducing the effects of the American leaf spot of coffee (Mycena citricolor). One of these isolates (GU11N) from the plant Randia grandifolia was identified as Daldinia eschscholtzii (Xylariales). Its antagonism mechanisms, effects, and chemistry against M. citricolor were investigated by analyzing its volatile profile alone and in the presence of the pathogen in contactless and dual culture assays. The experimental design involved direct sampling of agar plugs in vials for headspace (HS) and headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, we used ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) to identify nonvolatile compounds from organic extracts of the mycelia involved in the interaction. Results showed that more volatile compounds were identified using HS-SPME (39 components) than those by the HS technique (13 components), sharing only 12 compounds. Statistical tests suggest that D. eschscholtzii inhibited the growth of M. citricolor through the release of VOCs containing a combination of 1,8-dimethoxynapththalene and terpene compounds affecting M. citricolor pseudopilei. The damaging effects of 1,8-dimethoxynaphthalene were corroborated in an in vitro test against M. citricolor pseudopilei; scanning electron microscopy (SEM) photographs confirmed structural damage. After analyzing the UHPLC-HRMS/MS data, a predominance of fatty acid derivatives was found among the putatively identified compounds. However, a considerable proportion of features (37.3%) remained unannotated. In conclusion, our study suggests that D. eschscholtzii has potential as a biocontrol agent against M. citricolor and that 1,8-dimethoxynaphthalene contributes to the observed damage to the pathogen’s reproductive structures

The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440

Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 μM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16 %), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA