Arbuscular mycorrhizal fungi enhance monoterpene production in red clover (Trifolium pratense L.): a potential tool for pest control.

The possibility of modifying terpene production in plants is a defensive strategy that has been studied in conjunction with their biosynthetic pathways. A biotic factor such as Arbuscular Mycorrhizal Fungi (AMF) could modify terpene production in Trifolium pratense L. In this work, the enzymatic production of monoterpenes in Superqueli INIA cultivar with two AMF was evaluated via HeadSpace-Gas Chromatography (HS-GC). A significant increase of (S)-limonene was found in plants inoculated with Claroideoglomus claroideum as well as with the AMF mix (genera Scutellospora, Acaulospora and Glomus). Moreover, significant increases in other monoterpenes such as (-)-ß-pinene, myrcene, linalool, were observed. Results showed higher monoterpene production capacities in the Superqueli-INIA cultivar, suggesting the participation of monoterpene synthases (MTS). The significant rise of (S)-limonene in red clover plants inoculated with AMF suggests this strategy could be implemented in an agronomical manage for controlling the H. obscurus, the primary pest.

Synthesis and Antibacterial Activity of Manganese-Ferrite/Silver Nanocomposite Combined with Two Essential Oils.

The antimicrobial activity of metal nanoparticles obtained by biogenic routes has been extensively reported. However, their combined use with other antimicrobial formulations, such as essential oils, remains scarcely explored. In this work, a manganese-ferrite/silver nanocomposite (MnFe2O4/Ag-NC) was synthesized in a two-step procedure: first, MnFe2O4 nanoparticles were produced by a coprecipitation method, followed by in situ biogenic reduction of silver ions using Galega officinalis. MnFe2O4/Ag-NC was characterized using transmission electron microscopy (TEM), scanning electron microscopy equipped with an energy dispersive X-ray analyzer (SEM-EDX), and a vibrating sample magnetometer (VSM-SQUID). The antibacterial activity if MnFe2O4/Ag-NC was evaluated against Pseudomonas syringae by determining its minimum inhibitory concentration (MIC) in the presence of two essential oils: eucalyptus oil (EO) and garlic oil (GO). The fractional inhibitory concentration (FIC) was also calculated to determine the interaction between MnFe2O4/Ag-NC and each oil. The MIC of MnFe2O4/Ag-NC was eightfold reduced with the two essential oils (from 20 to 2.5 µg mL-1). However, the interaction with EO was synergistic (FIC: 0.5), whereas the interaction with GO was additive (FIC: 0.75). Additionally, a time-kill curve analysis was performed, wherein the MIC of the combination of MnFe2O4/Ag-NC and EO provoked a rapid bactericidal effect, corroborating a strong synergism. These findings suggest that by combining MnFe2O4/Ag-NC with essential oils, the necessary ratio of the nanocomposite to control phytopathogens can be reduced, thus minimizing the environmental release of silver.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Acetylcholinesterase inhibitory activity from Amaryllis belladonna growing in Chile: enzymatic and molecular docking studies.

Alkaloid profiles from Amaryllis belladonna plants collected in Chile were examined by GC-MS to assess their inhibitory activity on acetylcholinesterase (AChE) using in vitro and in silico methodologies. The alkaloid extract was roughly separated by column chromatography on silica gel. AChE inhibitory activities from extracts and purified alkaloids were tested by the Ellman method and a molecular docking study was performed to assess the interaction between AChE and purified alkaloids. Sixteen alkaloids were found from hexane and chloroform extracts, and three were isolated and identified as buphanidrine, acetylcaranine and lycorine. Chloroform extract showed the greatest AChE inhibitory activity with IC50 value 8.89 µg/mL, whereas buphanidrine exhibited the highest inhibitory activity, with IC50 value 17.56 µg/mL. Inhibition kinetics showed that buphanidrine acts as a mixed inhibitor and molecular docking supports this inhibition mechanism. Overall, our study supports the potential use of A. belladonna as an alkaloid source with AChE inhibitory activity.[Formula: see text].

Bioconversion of potato solid waste into antifungals and biopigments using Streptomyces spp.

Potato waste was processed and used as a sole substrate for simultaneously producing antifungals and biopigments using Streptomyces spp. Out of three different Streptomyces isolates, strain SO6 stood out due to its ability to produce antifungals against economically important fungal phytopathogens and intracellular biopigments using potato waste powders without additional nutrients. This strain also showed the potential to secrete a broad range of enzymes for fermentation of eight sugars that could be involved in potato waste bioconversion. The results of the fermentation assay indicated that Streptomyces sp. strain SO6 degrades potato wastes during submerged fermentation, diminishing total dry weight and increasing reducing sugars from 0.3 to 3.6 mg·mL-1 and total proteins from 70.6 to 187.7 µg·mL-1. The results showed that Streptomyces strain SO6 was able to convert the potato waste into 0.96 mg·g-1 of diffusible antifungals and 1.75 mg·g-1 of reddish-purple biopigments. On the contrary, an absence of pigment production was observed during the fermentation of the commercial medium used as reference. According to our results, replacement of commercial culture media with available low-cost agroindustrial wastes for producing bioactive chemicals is a real opportunity to enhance the Streptomyces pigment production and antibiotic sustainability with cost-competitiveness. To our knowledge, this is the first report on the simultaneous production of biopigments and diffusible antifungal antibiotics produced by Streptomyces spp. using potato solid waste as the sole nutrient source.

Galanthamine and other Amaryllidaceae related alkaloids are inhibitors of α7, α4β2 and α3β4 nicotinic acetylcholine receptors

Abstract Galanthamine is an Amaryllidaceae-derived acetylcholinesterase inhibitor used to treat memory impairment in Alzheimer's disease and vascular dementia. There is evidence that galanthamine, in addition to its effects on acetylcholinesterase, may enhance or inhibit brain nicotinic acetylcholine receptors, which could increase or decrease the therapeutic efficacy of galanthamine, respectively. Here, we evaluated the effects of galanthamine and two others Amaryllidaceae acetylcholinesterase inhibitors (haemanthamine and tazettine) analyzed by gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0 on the agonist responses of brain nicotinic acetylcholine receptors α7, α3β4, (α4)2(β2)3 and (α4)3(β2)2. Using nicotinic acetylcholine receptors expressed heterologously in Xenopus oocytes, in conjunction with two-electrode voltage clamping, we found that galanthamine inhibits the function of nicotinic acetylcholine receptors assayed through a mix competitive and non-competitevely. Nicotinic acetylcholine receptor α7 were significantly more sensitive to inhibition (17 ± 0.6 µM) than the heteromeric receptor, α3β4 (90 ± 3.4 µM). Neither haemanthamine nor tazettine were more potent than galanthamine.

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Rhodolirium andicola: a new renewable source of alkaloids with acetylcholinesterase inhibitory activity, a study from nature to molecular docking

ABSTRACT Acetylcholinesterase is an important target for control of neurodegenerative diseases causing cholinergic signaling deficit. Traditionally, galanthamine has been used as an Amaryllidaceae-derived acetylcholinesterase inhibitor, although new Amaryllidaceae plants could serve as source for better acetylcholinesterase inhibitors. Therefore, the objective of this study was to characterize the alkaloid composition from bulbs of Rhodolirium andicola (Poepp.) Traub, a native Chilean Amaryllidaceae specie, and assess their inhibitory activity on acetylcholinesterase by in vitro and in silico methodologies. Alkaloidal extracts from R. andicola exhibited an inhibitory activity with IC50 values between 11.25 ± 0.04 and 57.78 ± 1.92 µg/ml that included isolated alkaloid, galanthamine (2.3 ± 0.18 µg/ml), Additionally, 12 alkaloids were detected using gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0. To better understand the bioactivity of isolated compounds and alkaloidal extracts against acetylcholinesterase, a molecular docking approach was performed. Results suggested that alkaloids such as lycoramine, norpluvine diacetate and 6α-deoxy-tazettine expand the list of potential acetylcholinesterase inhibitors to not only galanthamine. The role of R. andicola as a source for acetylcholinesterase inhibitors is further discussed in this study.

Novel antimicrobial activity of a dichloromethane extract obtained from red seaweed Ceramium rubrum (Hudson) (Rhodophyta: Florideophyceae) against Yersinia ruckeri and Saprolegnia parasitica, agents that cause diseases in salmonids

Background Enteric red mouth disease and Saprolegniasis, which are caused by the bacteria Yersinia ruckeri and the oomycete Saprolegnia parasitica, respectively, are important illnesses that affect salmonid farming. Sanitary problems in farms are addressed by the prevention of disease outbreaks or by the treatment of diseases with chemicals. Environmental and governmental restrictions, toxicity and high treatment costs limit the use of drugs. Marine organisms, such as algae, sponges and corals, have developed an antimicrobial defense strategy based on the production of bioactive metabolites. Among these organisms, seaweeds offer a particularly rich source of potential new drugs. Hence, many pharmacologically active substances have been isolated from seaweeds. In the Ceramium genus, Ceramium rubrum has been emphasized by several authors for its antimicrobial properties. Based on this background, the present study focused on the antimicrobial activity of a lipophilic extract of C. rubrum on Y. ruckeri and S. parasitica. Results The alga, collected from the Pacific coast of Chile, underwent an ethanol extraction, and the concentrated extract was partitioned between water and dichloromethane. From the dichloromethane extract, fatty acids, fatty acid esters, one hydrocarbon and phytol were identified by Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antimicrobial study showed that the whole extract was more active than the individual components, which suggests a strong synergistic effect among the components. Conclusions These results may constitute a basis for promising future applied research that could investigate the use of C. rubrum seaweed as a source of antimicrobial compounds against fish pathogens.

Host selection and probing behavior of the poplar aphid Chaitophorus leucomelas (Sternorrhyncha: Aphididae) on two poplar hybrids with contrasting susceptibility to aphids.

Poplars are frequently attacked by aphids. The differential susceptibility of poplar hybrids to the aphid Chaitophorus leucomelas Koch (Sternorrhyncha: Aphididae) has been described, but the mechanism underlying this pattern is unknown. This work tested the hypothesis that poplar resistance to this aphid is associated with the presence of volatiles and secondary plant compounds that affect host selection and feeding behavior. This hypothesis was tested by studying the host choice and feeding behavior of C. leucomelas on two poplar hybrids with contrasting susceptibilities to this aphid ([Populus trichocarpa Torrey & Gray x Populus deltoides Bartram ex Marshall] x P. deltoides [TD x D], and [P. trichocarpa x Populus maximowiczii Henry] x [P. trichocarpa x P. maximowiczii] [TM x TM]). The results showed that C. leucomelas rejected leaves of the TM x TM hybrid and did not prefer odors from either hybrid. Electronic monitoring of the probing behavior of C. leucomelas suggested the involvement of antifeedant factors in the TM x TM hybrid. In addition, the chemical characterization of volatiles, epicuticular waxes, and internal phenols of leaves from both poplar hybrids revealed that TM x TM had a higher abundance of monoterpenes, sesquiterpenes, n-alkanes, and phenols. These results are discussed in terms of their contribution to poplar breeding programs aimed at enhancing insect resistance.

Chemical composition and antibacterial activity of Laureliopsis philippiana (Looser) essential oil / Composición química y actividad antibacteriana del aceite esencial de Laureliopsis philippiana (Looser)

Laureliopsis philippiana (Looser) is native evergreen specie from Chile and Argentina used in traditional medicine. In this study, chemical composition as well as its in vitro antibacterial activity against Escherichia coli, Enterobacter aerogenes, Staphylococcus epidermidis and Staphylococcus aureus of essential oil from leaves of this species was determinated. Chemical analysis by GC-MS resulted in the identification of 19 compounds representing 98.8 percent onoterpenes; linalool (32.3 percent) and eucalyptol (37.4 percent) were the main constituents. To evaluate the antibacterial activity disc diffusion method and broth dilution method were used. The essential oil exhibited inhibitory activity against Gram (-) and Gram (+) bacteria, whereas similar activity to essential oil was showed for linalool against E. aerogenes and S. epidermidis whereas linalool alone, achieves an inhibitory effect against E. aerogenes and S. epidermidis comparable to the essential oil.

Laureliopsis philippiana (Looser) es una especie siempre verde nativa de Chile y Argentina usado en medicina tradicional. En este estudio se determinó tanto la composición química del aceite esencial obtenido a partir de hojas de esta especie, así como su actividad antibacterial in vitro contra Escherichia coli, Enterobacter aerogenes, Staphylococcus epidermidis and Staphylococcus aureus. El análisis químico por GC-MS permitió la identificación de 19 compuestos, representando el 98,8 por ciento de la composición del aceite. Monoterpenos oxigenados, eucaliptol y linalol fueron los mayores constituyentes del aceite con un 37,4 por ciento y 32,3 por ciento respectivamente. Para evaluar la actividad antibacteriana se utilizaron los métodos de difusión en agar y dilución en caldo. El aceite esencial muestra actividad inhibitoria contra las bacterias Gram (-) y Gram (+) evaluadas, mientras que linalol por si solo logra un efecto inhibitorio comparable con el aceite esencial contra E. aerogenes y S. epidermidis mientras que el linalol por si solo, logra un efecto inhibitorio contra E. aerogenes y S. epidermidis comparable al del aceite esencial.

Cycloaspeptide A and pseurotin A from the endophytic fungus Penicillium janczewskii.

Penicillium janczewskii K. M. Zalessky was isolated as an endophytic fungus from the phloem of the Chilean gymnosperm Prumnopitys andina. When grown in liquid yeast extract-malt extract-glucose broth, the fungus produced two main secondary metabolites. The compounds were for the first time isolated from this species and identified by spectroscopic methods as pseurotin A and cycloaspeptide A. This is the first report on the production of cyclic peptides by endophytic fungi from Chilean gymnosperms. Pseurotin A and cycloaspeptide A presented low cytotoxicity towards human lung fibroblasts with IC50 > or = 1000 microM. Pseurotin A showed a moderate effect against the phytopathogenic bacteria Erwinia carotovora and Pseudomonas syringae, with IC50 values of 220 and 112 microg ml(-1), respectively.

Metabolites from Microsphaeropsis olivacea, an endophytic fungus of Pilgerodendron uviferum.

Seven compounds belonging to different structural skeletons were isolated from Microsphaeropsis olivacea grown in liquid and solid media. The enalin derivative 7-hydroxy-2,4-dimethyl-3(2H)-benzofuranone is reported for the first time, while additional spectroscopic information is provided for the acetates of botrallin and ulocladol. The activity of the isolated compounds was assessed towards the enzyme acetylcholinesterase (AChE) and their cytotoxicity against human lung fibroblasts. Graphislactone A and botrallin presented a moderate activity towards AChE, with IC50 of 8.1 and 6.1 microg/ml (27 and 19 microM, respectively). Under the same experimental conditions, the IC50 of the standard inhibitor galanthamine was 3 microg/ml. The cytotoxicity of both compounds was > 1000 and 330 microM, respectively. None of the compounds was promising as antibacterial or antifungic against phytopathogenic fungi and bacteria. Botrallin and graphislactone A were detected in the liquid potato-dextrose and yeast extract/malt extract/dextrose as well as on a solid substrate (rice). Butyrolactone I was obtained from the fungus growing on solid medium.

Secondary metabolite content in Fabiana imbricata plants and in vitro cultures.

A rapid in vitro propagation system leading to the formation of shoots, calli, roots, cell suspensions and plantlets was developed for the Andean medicinal plant Fabiana imbricata (Solanaceae). Massive propagation of shoots and roots was achieved by the temporary immersion system (TIS), morphogenesis and maintenance of cell suspensions by standard in vitro culture techniques. Oleanolic acid (OA), rutin, chlorogenic acid (CA) and scopoletin content in aerial parts of wild growing Fabiana imbricata plants as well as in plantlets regenerated in vitro, callus cultures, cell suspensions and biomass, obtained by the TIS system was assessed by HPLC. On a dry weight basis, the OA content in the aerial parts of the plant ranged between 2.26 and 3.47% while in vitro plantlets, callus and root cultures presented values ranging from not detected up to 0.14%. The rutin content of the samples presented a similar trend with maxima between 0.99 and 3.35% for the aerial parts of the plants to 0.02 to 0.20% for plantlets, 0.12% for cell suspensions and 0.28% for callus. Rutin was not detected in the roots grown by the TIS principle. The CA and scopoletin content in the aerial parts of F. imbricata ranged between 0.22-1.15 and < 0.01-0.55%, respectively. In the plantlets, the concentration of CA was 0.29 to 1.48% with scopoletin in the range 0.09 to 0.64% while in the callus sample, the CA and scopoletin content were 0.46 and 0.66%, respectively. A very different result was found in roots grown by TIS, where both OA and rutin were not detected and its main secondary metabolite, scopoletin was found between a range of 0.99 and 1.41% with CA between of 0.11 and 0.42%.