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.

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.

Trichoderma koningiopsis (Hypocreaceae) has the smallest mitogenome of the genus Trichoderma.

Introduction: Fungal mitogenomes exhibit remarkable variation in conformation, size, gene content, arrangement and expression, including their intergenic spacers and introns. Methods: The complete mitochondrial genome sequence of the mycoparasitic fungus Trichoderma koningiopsis was determined using the Illumina next-generation sequencing technology. We used data from our recent Illumina NGS-based project of T. koningiopsis genome sequencing to study its mitochondrial genome. The mitogenome was assembled, annotated, and compared with other fungal mitogenomes. Results: T. koningiopsis strain POS7 mitogenome is a circular molecule of 27,560 bp long with a GC content of 27.80%. It harbors the whole complement of the 14 conserved mitochondrial protein-coding genes (PCG) such as atp6, atp8, atp9, cox1, cox2, cox3, cob, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6, also found in the same gene order to other Hypocreales. The mitogenome also contains 26 transfer RNA genes (tRNAs), 5 of them with more than one copy. Other genes also present in the assembled mitochondrial genome are a small rRNA subunit and a large rRNA subunit containing ribosomal protein S3 gene. Despite the small genome size, two introns were detected in the T. koningiopsis POS7 mitogenome, one of them in cox3 gene and the other in rnl gene, accounting 7.34% of this mitogenome with a total size of 2,024 bp. A phylogenetic analysis was done using the 14 PCGs genes of T. koningiopsis strain POS7 mitogenome to compare them with those from other fungi of the Subphyla Pezizomycotina and Saccharomycotina. T. koningiopsis strain POS7 was clustered together with other representatives of Trichoderma lineage, within the Hypocreales group, which is also supported by previous phylogenetic studies based on nuclear markers. Discussion: The mitochondrial genome of T. koningiopsis POS7 will allow further investigations into the taxonomy, phylogenetics, conservation genetics, and evolutionary biology of this important genus as well as other closely related species.

Are dark septate endophytes an ancestral ecological state in the evolutionary history of the order Chaetothyriales?

Dark septate endophytes (DSE) are pigmented fungi that colonize plant roots. They represent a morpho-functional status composed of many species belonging to the phylum Ascomycota, distributed in different orders. The order Chaetothyriales has representatives with diverse lifestyles, among which the rock-inhabiting one has been proposed to be the ancestral ecological character state. However, all taxa have the phenotypic characteristic of being highly melanized. This trait has been considered relevant in most Chaetothyriales because it allows them to tolerate extreme or toxic environmental conditions. In the present study, aiming to reconstruct the evolutionary history of this order, we analyzed the contribution of the DSE habit to the diversification of the Chaetothyriales. We also report the distribution of the DSE habit among the main families and/or clades within the order. Our results suggest that DSE had a key position in the evolution of the order Chaetothyriales, both as an ancestral ecological character and as a character from which other specialized forms such as Domatium probably derived.

Mushroom Production in the Southern Cone of South America: Bioeconomy, Sustainable Development and Its Current Bloom.

A Sustainable Development Goals (SDGs) based analysis is presented here for business development of the production of edible and medicinal mushrooms using agro-wastes in the Southern Cone of South America. This circular economy approach using edible and medicinal mushroom production on lignocellulosic residues is discussed by analysing both its advantages and drawbacks. Among its main benefits, it is notable that mushroom cultivation using lignocellulosic residues promotes innovation aimed at environmental sustainability, facilitating diversification of the labour supply and the transfer of science to the socio-cultural sphere, which also increases the availability of healthy foods. However, there are some bottlenecks in the process, such as the continuous supply chain of substrates for fungal growth, the lack of equipment and infrastructure for the implementation of cultivation systems in extreme habitats, as well as authorization requirements and other limitations related to a non-fungiphilic culture society. Therefore, this chapter tries to provide key tools for establishing sustainable guidelines for the procurement of local healthy food and other products derived from mushroom cultivation using agricultural residues in the region, which might bloom due to an SDGs-based circular economy approach.

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.

Endomycobiome associated with females of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae): A metabarcoding approach.

A metabarcoding approach was performed aimed at identifying fungi associated with Delphacodes kuscheli (Hemiptera: Delphacidae), the main vector of "Mal de Río Cuarto" disease in Argentina. A total of 91 fungal genera were found, and among them, 24 were previously identified for Delphacidae. The detection of fungi that are frequently associated with the phylloplane or are endophytes, as well as their presence in digestive tracts of other insects, suggest that feeding might be an important mechanism of their horizontal transfer in planthoppers. This study draws the baseline for future research regarding mutualistic associations present in D. kuscheli as well as their physiological role in the life cycle of this important pest that might lead to developing new management strategies to keep insects populations under control.

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.

Fungal Mitogenomes: Relevant Features to Planning Plant Disease Management.

Mitochondrial genomes (mt-genomes) are characterized by a distinct codon usage and their autonomous replication. Mt-genomes encode highly conserved genes (mt-genes), like proteins involved in electron transport and oxidative phosphorylation but they also carry highly variable regions that are in part responsible for their high plasticity. The degree of conservation of their genes is such that they allow the establishment of phylogenetic relationships even across distantly related species. Here, we describe the mechanisms that generate changes along mt-genomes, which play key roles at enlarging the ability of fungi to adapt to changing environments. Within mt-genomes of fungal pathogens, there are dispensable as well as indispensable genes for survival, virulence and/or pathogenicity. We also describe the different complexes or mechanisms targeted by fungicides, thus addressing a relevant issue regarding disease management. Despite the controversial origin and evolution of fungal mt-genomes, the intrinsic mechanisms and molecular biology involved in their evolution will help to understand, at the molecular level, the strategies for fungal disease management.

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.

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.

Perfiles de utilización de sustratos carbonados de Cladorrhinum (Ascomycota) / Carbon-substrate utilization profiles by Cladorrhinum (Ascomycota)

Abstract 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.

Resumen Los hongos del género Cladorrhinum (Ascomycota) son agentes prometedores en el biocontrol de fitopatógenos, la promoción del crecimiento de las plantas y la producción de enzimas con aplicación tecnológica. En este trabajo se analizaron comparativamente las habilidades de 5 cepas nativas pertenecientes a las especies Cladorrhinum samala y Cladorrhinum bulbillosum con cepas de referencia del mismo género. Se usaron 95 fuentes individuales de carbono, disponibles en microplacas de Biolog® FF system. Aunque la mayoría de las cepas utilizaron principalmente carbohidratos solubles, el perfil metabólico fue altamente dependiente de cada aislamiento y reveló variabilidad fisiológica intraespecífica en las especies de Cladorrhinum.

Effects of the fungus Pestalotiopsis maculans (Ascomycota: Amphisphaeriales) on the gametophytic development of the fern Lygodium venustum (Lygodiaceae)

Lygodium Sw. is the only genus of the family Lygodiaceae, with around 25 to 40 species inhabiting mostly tropical regions worldwide. In Argentina two species develop; one of them, L. venustum, grows in the Northeast of the country. Some species of Lygodium are invasive whereas medicinal uses have been reported for some taxa. As part of a project to ascertain on the reproductive biology of native ferns in Argentina, a fungus was found during in vitro spore culture of L. venustum. Dark brown spots and eruptive pustules were also observed in fertile leaves. Thus, emerged as objectives of this work: 1) to retrieve and identify the fungus from the sporophyte and from the spores cultures, 2) to inquire on the impact of the fungus on gametophyte generation, and 3) to perform experiments with untreated spores to demonstrate that the fungus is associated to the spores collected from infested sporophytes. Phytopathological techniques were applied in order to isolate the fungus and then to achieve monosporic cultures. The morpho-biometric features of the conidia corresponded with those of Pestalotiopsis maculans (Corda) Nag Raj [= Pestalotiopsis guepinii (Desm.) Steyaert] (Amphisphaeriales, Ascomycota). Re-inoculation of conidia on healthy gametophytes conducted to their necrosis and death. The percentages of spore germination were initially higher in unsterilized cultures, suggesting that disinfection protocol may have affected spore germination. This is the first report of Pestalotiopsis maculans on the sporophytes of Lygodium venustum. The information that emerges from our work could be useful for the biocontrol of other species of Lygodium, considered weeds in other regions of the world.

Efectos del hongo Pestalotiopsis maculans (Ascomycota: Amphisphaeriales) en el desarrollo gametofítico del helecho Lygodium venustum (Lygodiaceae). Lygodium Sw. es el único género de la familia Lygodiaceae, con alrededor de 25 a 40 especies que habitan en su mayoría en regiones tropicales del mundo. En Argentina se desarrollan dos especies; uno de ellas, L. venustum, crece en el noreste del país. Algunas especies de Lygodium son invasivas, mientras que para algunos taxones se han reportado usos medicinales. Como parte de un proyecto para indagar sobre la biología reproductiva de los helechos nativos en Argentina, se encontró un hongo durante el cultivo in vitro de esporas de L. venustum. Asimismo, se observaron manchas marrones oscuras y pústulas eruptivas en hojas fértiles. Así, surgieron como objetivos de este trabajo: 1) recuperar e identificar el hongo presente en el esporofito y en los cultivos de esporas, 2) investigar el impacto del hongo en la generación gametofítica y 3) realizar experimentos con esporas no tratadas para demostrar que el hongo está asociado a las esporas recolectadas de esporofitos infestados. Se aplicaron técnicas fitopatológicas para aislar el hongo y luego lograr cultivos monospóricos. Las características morfo-biométricas de los conidios correspondieron a las de Pestalotiopsis maculans (Corda) Nag Raj [= Pestalotiopsis guepinii (Desm.) Steyaert] (Amphisphaeriales, Ascomycota). La re-inoculación de conidios en gametofitos sanos condujo a su necrosis y muerte. Los porcentajes de germinación de esporas fueron inicialmente más altos en los cultivos no esterilizados, lo que sugiere que el protocolo de desinfección pudo haber afectado la germinación de las esporas. Este es el primer reporte de Pestalotiopsis maculans sobre los esporofitos de Lygodium venustum. La información que surge de nuestro trabajo podría ser útil para el control biológico de otras especies de Lygodium, consideradas malezas en otras regiones del mundo.

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.

Assemblage of dematiaceous and Ingoldian fungi associated with leaf litter of decomposing Typha latifolia L. (Typhaceae) in riverine wetlands of the Pampean plain (Argentina) exposed to different water quality.

The purpose of this study was to analyze the rate of sporulation, richness, and spore diversity of dematiaceous and Ingoldian fungi colonizing Typha latifolia leaves during a 40-day period of decomposition, as well as the loss of mass in Typha latifolia, in four riverine wetlands of Pampean plain (Argentina) with different water quality. Higher sporulation rates, richness, and diversity of the fungi as well as loss of mass of the leaves that they colonized were associated with lower water quality. Anguilospora longissima, Arthrinium sp., Margaritispora aquatica, and Tricellula botryosa were dominant taxa. Redundancy analysis showed two fungal assemblages related to different environmental conditions. One assemblage was related to higher nutrient levels and higher temperature, characterized mainly by dematiaceous fungi. The other assemblage was related to higher levels of pH and dissolved oxygen, which was mainly represented by Ingoldian fungi. The results obtained in our study demonstrated the link between these fungal assemblages and changes in water quality, revealing their potential as indicators of environmental changes in rivers exposed to different types of land use.

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.

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.

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.

Enhancing laccase production by white-rot fungus Funalia floccosa LPSC 232 in co-culture with Penicillium commune GHAIE86.

To obtain enzymatic preparations with higher laccase activity levels from Funalia floccosa LPSC 232, available for use in several applications, co-cultures with six filamentous microfungi were tested. A laccase non-producing soil fungus, identified as Penicillium commune GHAIE86, showed an outstanding ability to increase laccase activity (3-fold as compared to that for monoculture) when inoculated in 6-day-old F. floccosa cultures. Maximum laccase production with the F. floccosa and P. commune co-culture reached 60 U/mL, or twice that induced by chemical treatments alone. Our study demonstrated that co-culture with soil fungi might be a promising method for improving laccase production in F. floccosa. Although the enhancement of laccase activity was a function of P. commune inoculation time, two laccase isoenzymes produced by F. floccosa remained unchanged when strains were co-cultured. These data are compatible with the potential of F. floccosa in agricultural applications in soil, whose enzyme machinery could be activated by soil fungi such as P. commune.