Biocidal and antibiofilm activities of arginine-based surfactants against Candida isolates.

Amino-acid-based surfactants are a group of compounds that resemble natural amphiphiles and thus are expected to have a low impact on the environment, owing to either the mode of surfactant production or its means of disposal. Within this context, arginine-based tensioactives have gained particular interest, since their cationic nature-in combination with their amphiphilic character-enables them to act as broad-spectrum biocides. This capability is based mainly on their interactive affinity for the microbial envelope that alters the latter's structure and ultimately its function. In the work reported here, we investigated the efficiency of Nα-benzoyl arginine decyl- and dodecylamide against Candida spp. to further our understanding of the antifungal mechanism involved. For the assays, both a Candida albicans and a Candida tropicalis clinical isolates along with a C. albicans-collection strain were used as references. As expected, both arginine-based compounds proved to be effective against the strains tested through inhibiting both the planktonic and the sessile growth. Furthermore, atomic force microscopy techniques and lipid monolayer experiments enabled us to gain insight into the effect of the surfactant on the cellular envelope. The results demonstrated that all the yeasts treated exhibited changes in their exomorphologic structure, with respect to alterations in both roughness and stiffness, relative to the nontreated ones. This finding-in addition to the amphiphiles' proven ability to insert themselves within this model fungal membrane-could explain the changes in the yeast-membrane permeability that could be linked to viability loss and mixed-vesicle release.

Studies on aquatic fungi in Dikarya: a review of the literature from Southern Cone of South America.

A bibliographic analysis was carried out to update the state of knowledge about aquatic fungi belonging to the subkingdom Dikarya in the Southern Cone of South America. The exhaustive search resulted in 38 articles reported. These papers correspond to those on taxonomic, ecological and biogeographic topics and include studies from lotic environments of the temperate ecoregions of Chile and Argentina. A total of 325 aquatic fungal taxa were reported, of which 318 belong to the phylum Ascomycota and 7 to the phylum Basidiomycota. According to the subgroups of these aquatic fungi 17 taxa were aero-aquatic, 199 facultative and 109 Ingoldian fungi. Regarding the methodologies, in these studies the information was obtained mainly by using lignocellulosic substrates such as leaf litter and wood as fungal source and wet chamber traditional working technique. However, more studies are still needed using other few-reported perspectives for the region such as ecological and molecular approaches as well as analyses of water environments belonging to unexplored biomes. This information can contribute to a better understanding of aquatic fungal communities and their role in ecosystems of the Southern Cone of South America.

Mycobiota on exoskeleton debris of Neohelice granulata in an alkaline-sodic salt marsh: in vitro enzyme ability at different temperatures and pH.

This study analysed the mycobiota on exoskeleton debris of the crab Neohelice granulata collected from an alkaline salt marsh and assessed the in vitro enzyme ability of selected isolates at different temperatures and pH. Exoskeleton fragments were incubated in moist chambers on paper and on agar medium. Growth and enzyme ability of selected fungi were also evaluated in agar media with 0.5 % casein, 1% Tween®20, and Chitin-Azure® by the production of a halo/growth ratio. We identified 22 fungal species using both methods. Since the two isolation methods added information to one another, both ones are necessary to recover the cultivable mycobiota associated with the exoskeleton debris. All fungi showed greater levels of enzyme activity in alkaline than acid medium with Tween®20. The halo diameter on casein and chitin varied according to the fungal isolate and pH. Most fungi had a larger halo at 4°C than at the other temperatures tested. Clonostachys rosea showed the greatest activity in all media at 4ºC. We conclude that exoskeletons of the N. granulata are a source of fungi able to produce enzyme activities that show differences upon incubation conditions to which they are cultivated such as ones including specific temperatures and pH values.

Biotransformation of grape pomace from Vitis labrusca by Peniophora albobadia LPSC # 285 (Basidiomycota).

Grape pomace from Vitis labrusca is an important sub-product of the "American table wine" industry. It is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. We analyzed the ability of five white-rot fungi to transform this residue in-vitro. Mass loss and phenol removal in grape pomace treated with each fungus were compared after 30-day solid-state fermentation. Since Peniophora albobadia isolate LPSC 285 was the fungus that showed the highest degradative ability and the lowest free phenol levels in the residue transformed, we selected this fungus to monitor its effect on this residue after 30, 60, and 90 days of incubation. We analyzed mass loss of the residue caused by the fungus activity and its chemical changes using Fourier transform infrared spectroscopy. After 90 days of incubation, Peniophora albobadia isolate LPSC 285 reduced grape pomace mass by 20.48%, which was associated with degradation of polysaccharides and aromatic structures. We concluded that Peniophora albobadia LPSC # 285 isolate is a promising fungus to transform grape pomace from Vitis labrusca under solid-state fermentation conditions.

Laccases: structure, function, and potential application in water bioremediation.

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

Cationic surfactants as antifungal agents.

Fungi-in being responsible for causing diseases in animals and humans as well as environmental contaminations in health and storage facilities-represent a serious concern to health security. Surfactants are a group of chemical compounds used in a broad spectrum of applications. The recently considered potential employment of cationic surfactants as antifungal or fungistatic agents has become a prominent issue in the development of antifungal strategies, especially if such surface-active agents can be synthesized in an eco-friendly manner. In this review, we describe the antifungal effect and the reported mechanisms of action of several types of cationic surfactants and also include a discussion of the contribution of these surfactants to the inhibition of yeast-based-biofilm formation. Furthermore, the putative mechanism of arginine-based tensioactive compounds as antifungal agents and their applications are also analyzed.

Carbon-substrate utilization profiles by Cladorrhinum (Ascomycota).

Fungi from the genus Cladorrhinum (Ascomycota) are promising agents in the biocontrol of phytopathogens, in the promotion of plant growth, and in the production of enzymes with technological application. We analyzed comparatively the ability of 5 native strains of Cladorrhinum samala and Cladorrhinum bulbillosum with reference strains belonging to the same genus. We used 95 individual carbon sources available in microplates from the Biolog® FF system. Although most of the strains mainly used soluble carbohydrates, the metabolic profile was highly dependent upon each isolate and it revealed intraspecific physiological variability in Cladorrhinum species.

A Survey on Occurrence of Cladosporium fulvum Identifies Race 0 and Race 2 in Tomato-Growing Areas of Argentina.

The presence of Cladosporium fulvum (syn. Passalora fulva), causal agent of tomato leaf mold, was confirmed in the two main greenhouse-production areas for tomato in Argentina. Using both morphological characters and internal transcribed spacer sequencing, we confirmed the presence of physiological races of this pathogen. A diagnostic multiplex polymerase chain reaction (PCR) was also developed, using primers derived from C. fulvum avirulence (Avr) genes. In all, 20 isolates of Cladosporium spp. were obtained as monospore cultures and 12 were identified as C. fulvum. By this method, we showed that, of these 12 isolates, 5 were race 0 (carrying functional Avr2, Avr4, Avr4E, and Avr9 genes) and 7 were race 2 (lacking the Avr2 gene). Race identity was confirmed by testing their virulence on a set of tomato differentials carrying different Cf resistance genes. All Avr genes could be amplified in single or multiplex PCR using DNA isolated from in vitro grown monospore cultures but only three Avr could be amplified when genomic DNA was isolated from C. fulvum-infected necrotic leaf tissue.

Coriolopsis rigida, a potential model of white-rot fungi that produce extracellular laccases.

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect remain to be elucidated. C. rigida's ability to degrade lignin and lignin-related compounds and the capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied processes.

Dark Pigments in Entomopathogenic Fungal Microsclerotia: Preliminary Evidence of a 1,8-Dihydroxynaphthalene-melanin-like Compound in Metarhizium robertsii.

Metarhizium robertsii microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of M. robertsii microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained M. robertsii propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, M. robertsii microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.

Cladosporium cladosporioides LPSC 1088 produces the 1,8-dihydroxynaphthalene-melanin-like compound and carries a putative pks gene.

Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi.

Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina.

Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4 degreeC, 12 degreeC and 24 degreeC), on different agar media supplemented with the following specific substrates: chitin azure, Tween 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4 degree C, 12 degree C and 24 degree C). The suspension was adjusted to a concentration of 1x10(7) conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24 degreeC), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween 20 at the lower two incubation temperatures of 4 degreeC or 12 degreeC. In accordance with the results on colony diameters, the fungus revealed an ability to degrade casein, chitin derivatives, Tween 20, and urea as evidenced by the appearance of a halo around the fungal colony. Because of its origin and temperature tolerance, this Argentine strain has great potential for use as a biocontrol agent for insect pest control in cold and temperate environments.

Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production.

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

Fungal melanins that deteriorate paper cultural heritage: An overview.

Paper-based works of art and documents of cultural importance kept in museums and libraries can show notorious signs of deterioration, including foxing stains, caused by fungal colonization. Some of the main chromophore agents of fungal origin that deteriorate paper and therefore affect paper cultural heritage both aesthetically and structurally are the group of pigments called melanins. Thus, knowledge of the diversity and features of fungal melanins and of the melanization pathways of fungi growing on paper is key to removing these pigments from paper-based works of cultural importance. This review provides an approach about the current knowledge of melanins synthesized by paper-colonizing fungi, their localization in the fungal structures, and their role in the deterioration of paper. This knowledge might contribute to developing new, effective, and sustainable strategies of restoration and conservation of historical documents and works of art based on paper.

Changes in microbial communities during phytoremediation of contaminated soil with phenanthrene.

Although polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that affect negatively soils biology, several strategies lead to their removal such as the phytoremediation. In order to assess the potential of phytoremediation using "alfalfa" Medicago sativa as a strategy to reduce the phenanthrene on the soil, we analyzed the structure and dynamic of the microbial communities of a microcosm soil artificially contaminated with phenanthrene (2000 ppm), which was exposed to the plants. At different incubation times (7, 14, 21, 28, 42, and 56 days), a soil sample was taken from each microcosm and the residual amount of phenanthrene was quantified. Dehydrogenase activity and the count of fungi and bacteria were also estimated. Bacterial communities were characterized using PCR-DGGE, Shannon and Weaver's indexes, multivariate analysis, and rarefaction curves. It was found that phytoremediation treatment was associated with a higher richness and bacterial diversity compared with those on control soil. Although an OTUs (Operational Taxonomic Unit) succession over time was detected in both treatments, bacterial richness and diversity were conditioned by the phenanthrene concentration available and also dependent on the treatment, which were associated to different bacterial communities. In this study, phytoremediation treatment reduced the content of phenanthrene in the soil after 56 days to a 0.45% compared with the control treatment, which only reached to 4.25%. This preliminary work suggests the promoting activity of "alfalfa" plants, through rhizodegradation, to remove in soil PAHs, as well as its relevance in the activation of different ecological processes mediated by soil microorganisms.

Pseudocercospora griseola causing angular leaf spot on Phaseolus vulgaris produces 1,8-dihydroxynaphthalene-melanin.

Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two major groups have been defined, "Andean" (P. griseola f. griseola) and "Mesoamerican" (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 +/- 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 +/- 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment, a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum. The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola.

Fungal transformation and reduction of phytotoxicity of grape pomace waste.

Grape pomace (GP) from Vitis labrusca, the main byproduct from "American table wine" production, is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. In this work, transformation of grape pomace using a steam pretreatment followed by incubation of GP during a 90-day period with six different fungi were evaluated. Several fungi tested reduced the phytotoxicity of water-soluble fraction (WSFd) from steam-pretreated GP after 90 days' incubation to lettuce and tomato seeds. U. botrytis caused the largest effective phytotoxicity reduction of WSFd (used in the concentration range of 10-1.25% p/v) and was the only fungus causing the removal of monoaromatic compounds. Therefore, this procedure with U. botrytis effectively reduces the availability of phytotoxic monoaromatic compounds in GP, which opens a way for the development of guidelines for the management of these wastes and their potential use as organic amendments in agricultural soil.

Frequency, abundance and distribution of zoosporic organisms from Las Cañas stream (Buenos Aires, Argentina).

Zoosporic organisms are common inhabitants of aquatic environments; however there are few ecological studies made for Argentinean streams. In this contribution the taxonomic composition of zoosporic organisms from a stream and their abundance, frequency and diversity on cellulosic baits were analyzed. Samples of water and floating organic matter (vegetable debris) were taken at four dates and different environmental variables (temperature, dissolved oxygen and nutrient concentrations) were measured. Twenty-one taxa were recovered with the baiting technique. Physicochemical fluctuations affected the structure of the studied community; in spring the greatest species richness was related to high nutrient levels whereas in winter the greatest abundance and diversity was related to low water temperature, nutrient levels and well oxygenated conditions. aquatic environment,

Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes.

Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

Melanins from two selected isolates of Pseudocercospora griseola grown in-vitro: Chemical features and redox activity.

Pseudocercospora griseola is the causal agent of Angular Leaf Spot (ALS), a disease of common bean. Due to its coevolution with beans, two major groups have been defined, "Andean" (P. griseola f. griseola) and "Mesoamerican" (P. griseola f. mesoamericana). The aim of this study was to characterize the dark pigment, melanin, synthetized by a selected isolate of each genic group of P. griseola when grown on Potato-dextrose broth. P. griseola f. griseola isolate S3b and P. griseola f. mesoamericana isolate T4 produced 1.7 ±â€¯0.6 and 4.1 ±â€¯0.9 mg of melanin per g of dry biomass, respectively. Although both melanins possessed similar UV-visible absorption spectroscopic pattern, P. griseola f. mesoamericana isolate T4 melanin had a lower UV-visible absorption, higher reducing activity and metal chelating ability than melanin from P. griseola f. griseola isolate S3b. However, when the size of the sample was 10 mg S3b melanin had a higher content of free phenolic groups. Furthermore, cell wall polysaccharides modified in melanin the availability of active phenolic groups, which was dependent on the fungal isolate and the size of the sample. Therefore, the amount and chemical features of melanin as well as its deposition in mycelium walls within isolates is different, which might explain the different pigmentation and physiological behaviours of these representatives of the two groups of P. griseola.