Exploring the infiltrative and degradative ability of Fusarium oxysporum on polyethylene terephthalate (PET) using correlative microscopy and deep learning.

Managing the worldwide steady increase in the production of plastic while mitigating the Earth's global pollution is one of the greatest challenges nowadays. Fungi are often involved in biodegradation processes thanks to their ability to penetrate into substrates and release powerful catabolic exoenzymes. However, studying the interaction between fungi and plastic substrates is challenging due to the deep hyphal penetration, which hinders visualisation and evaluation of fungal activity. In this study, a multiscale and multimodal correlative microscopy workflow was employed to investigate the infiltrative and degradative ability of Fusarium oxysporum fungal strain on polyethylene terephthalate (PET) fragments. The use of non-destructive high-resolution 3D X-ray microscopy (XRM) coupled with a state-of-art Deep Learning (DL) reconstruction algorithm allowed optimal visualisation of the distribution of the fungus on the PET fragment. The fungus preferentially developed on the edges and corners of the fragment, where it was able to penetrate into the material through fractures. Additional analyses with scanning electron microscopy (SEM), Raman and energy dispersive X-ray spectroscopy (EDX) allowed the identification of the different phases detected by XRM. The correlative microscopy approach unlocked a more comprehensive understanding of the fungus-plastic interaction, including elemental information and polymeric composition.

Interaction between a Martian Regolith Simulant and Fungal Organic Acids in the Biomining Perspective.

The aim of this study was to evaluate the potential of Aspergillus tubingensis in extracting metals from rocks simulating Martian regolith through biomining. The results indicated that the fungal strain produced organic acids, particularly oxalic acid, in the first five days, leading to a rapid reduction in the pH of the culture medium. This acidic medium is ideal for bioleaching, a process that employs acidolysis and complexolysis to extract metals from rocks. Additionally, the strain synthesized siderophores, molecules capable of mobilizing metals from solid matrices, as verified by the blue CAS colorimetric test. The secretion of siderophores in the culture medium proved advantageous for biomining. The siderophores facilitated the leaching of metal ions, such as manganese, from the rock matrix into the acidified water solution. In addition, the susceptibility of the Martian regolith simulant to the biomining process was assessed by determining the particle size distribution, acid composition after treatment, and geochemical composition of the rock. Although the preliminary results demonstrate successful manganese extraction, further research is required to optimize the extraction technique. To conclude, the A. tubingensis strain exhibits promising abilities in extracting metals from rocks through biomining. Its use could prove useful in future in situ mining operations and environmental remediation efforts. Further research is required to optimize the process and evaluate its feasibility on a larger scale.

Antarctic Soil Metabolomics: A Pilot Study.

In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the "gold standard" for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.

The Analysis of the Mycobiota in Plastic Polluted Soil Reveals a Reduction in Metabolic Ability.

Plastic pollution is a growing environmental issue that results in its accumulation and persistence in soil for many decades, with possible effects on soil quality and ecosystem services. Microorganisms, and especially fungi, are a keystone of soil biodiversity and soil metabolic capacity. The aim of this research was to study soil fungal biodiversity and soil microbial metabolic profiles in three different sites in northern Italy, where macro- and microplastic concentration in soil was measured. The metabolic analyses of soil microorganisms were performed by Biolog EcoPlates, while the ITS1 fragment of the 18S ribosomal cDNA was used as a target for the metabarcoding of fungal communities. The results showed an intense and significant decrease in soil microbial metabolic ability in the site with the highest concentration of microplastics. Moreover, the soil fungal community composition was significantly different in the most pristine site when compared with the other two sites. The metabarcoding of soil samples revealed a general dominance of Mortierellomycota followed by Ascomycota in all sampled soils. Moreover, a dominance of fungi involved in the degradation of plant residues was observed in all three sites. In conclusion, this study lays the foundation for further research into the effect of plastics on soil microbial communities and their activities.

Iridoids Isolation from a Phytochemical Study of the Medicinal Plant Teucrium parviflorum Collected in Iraqi Kurdistan.

Herbal medicines are still widely practiced in Kurdistan Region-Iraq, especially by people living in villages on mountainous regions. Among plants belonging to the genus Teucrium (family Lamiaceae), which are commonly employed in the Kurdish traditional medicine, we have analyzed, for the first time, the methanol and aqueous methanol extracts of T. parviflorum aerial parts. The plant is mainly used by Kurds to treat jaundice, liver disorders and stomachache. We aimed to determine the phytochemical profile of the extracts and the structures of the main components, so to provide a scientific rationale for the ancient use of the plant in the ethno-pharmacological field. TLC analysis of the two extracts on silica gel and reversed phase TLC plates, using different visualization systems, indicated similar contents and the presence of phenolics, flavonoids, terpenoids and sugars. The chlorophyll-free extracts exhibited weak/no antimicrobial activities against a panel of bacteria (MICs = 800-1600 µg/mL) and fungal strains (MICs ≥ 5 mg/mL). At the concentration of 600 µg/mL, the methanol extract showed moderate antiproliferative effects against A549 and MCF-7 cancer cell lines in the MTS assay. Moreover, both extracts exhibited a significant dose-dependent free radical scavenging action against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 = 62.11 and 44.25 µg/mL, respectively). In a phytochemical study, a high phenolic content (77.08 and 81.47 mg GAE/g dry extract, respectively) was found in both extracts by the Folin-Ciocalteu assay. Medium pressure liquid chromatographic (MPLC) separation of the methanol extract on a reversed phase cartridge eluted with a gradient of MeOH in H2O, afforded two bioactive iridoid glucosides, harpagide (1) and 8-O-acetylharpagide (2). The structures of 1 and 2 were established by spectral data, chemical reactions, and comparison with the literature. Interestingly, significant amounts of hepatotoxic furano neo-clerodane diterpenoids, commonly occurring in Teucrium species, were not detected in the extract. The wide range of biological activities reported in the literature for compounds 1 and 2 and the significant antiradical effects of the extracts give scientific support to the traditional use in Iraqi Kurdistan of T. parviflorum aerial parts for the preparation of herbal remedies.

Fungal Enzymes Involved in Plastics Biodegradation.

Plastic pollution is a growing environmental problem, in part due to the extremely stable and durable nature of this polymer. As recycling does not provide a complete solution, research has been focusing on alternative ways of degrading plastic. Fungi provide a wide array of enzymes specialized in the degradation of recalcitrant substances and are very promising candidates in the field of plastic degradation. This review examines the present literature for different fungal enzymes involved in plastic degradation, describing their characteristics, efficacy and biotechnological applications. Fungal laccases and peroxidases, generally used by fungi to degrade lignin, show good results in degrading polyethylene (PE) and polyvinyl chloride (PVC), while esterases such as cutinases and lipases were successfully used to degrade polyethylene terephthalate (PET) and polyurethane (PUR). Good results were also obtained on PUR by fungal proteases and ureases. All these enzymes were isolated from many different fungi, from both Basidiomycetes and Ascomycetes, and have shown remarkable efficiency in plastic biodegradation under laboratory conditions. Therefore, future research should focus on the interactions between the genes, proteins, metabolites and environmental conditions involved in the processes. Further steps such as the improvement in catalytic efficiency and genetic engineering could lead these enzymes to become biotechnological applications in the field of plastic degradation.

Soil Fungal Diversity of the Aguarongo Andean Forest (Ecuador).

Fungi represent an essential component of ecosystems, functioning as decomposers and biotrophs, and they are one of the most diverse groups of Eukarya. In the tropics, many species are unknown. In this work, high-throughput DNA sequencing was used to discover the biodiversity of soil fungi in the Aguarongo forest reserve, one of the richest biodiversity hotspots in Ecuador. The rDNA metabarcoding analysis revealed the presence of seven phyla: Ascomycota, Basidiomycota, Mortierellomycota, Mucoromycota, Glomeromycota, Chytridiomycota, and Monoblepharomycota. A total of 440 identified species were recorded. They mainly belonged to Ascomycota (263) and Basidiomycota (127). In Mortierellomycota, 12 species were recorded, among which Podila verticillata is extremely frequent and represents the dominant species in the entire mycobiota of Aguarongo. The present research provides the first account of the entire soil mycobiota in the Aguarongo forest, where many fungal species exist that have strong application potential in agriculture, bioremediation, chemical, and the food industry. The Aguarongo forest hides a huge number of unknown fungal species that could be assessed, and its protection is of the utmost importance.

The Mycobiota of High Altitude Pear Orchards Soil in Colombia.

In Colombia, the cultivation of deciduous fruit trees such as pear is expanding for socio-economic reasons and is becoming more and more important for the local population. Since organized cultivation is slowly replacing sustenance cultivation, scientific information on the present agro-environment is needed to proceed in this change in an organic and environmentally friendly way. In particular, this study is an accurate description of the mycobiota present in the bulk soil of two different high altitude pear orchards in the Colombian Andes. The metabarcoding of soil samples allowed an in-depth analysis of the whole fungal community. The fungal assemblage was generally dominated by Ascomycota and secondly by Mortierellomycota. As observed in other studies in Colombia, the genus Mortierella was found to be especially abundant. The soil of the different pear orchards appeared to host quite different fungal communities according to the soil physico-chemical properties. The common mycobiota contained 35 fungal species, including several species of Mortierella, Humicola, Solicoccozyma and Exophiala. Moreover, most of the identified fungal species (79%) were recorded for the first time in Colombian soils, thus adding important information on soil biodiversity regarding both Colombia and pear orchards.

Diversity of Mycobiota Associated with the Cereal Cyst Nematode Heterodera filipjevi Originating from Some Localities of the Pannonian Plain in Serbia.

Cereals, particularly wheat, are staple food of the people from the Balkans, dating back to the Neolithic age. In Serbia, cereals are predominantly grown in its northern part between 44° and 45.5° N of the Pannonian Plain. One of the most economically important nematodes on wheat is the cereal cyst nematode, Heterodera filipjevi. Cysts of H. filipjevi survive in soil for years and shelter a large number of microorganisms. The aims of this study were to investigate the diversity of mycobiota associated with the cereal cyst nematode H. filipjevi, to infer phylogenetic relationships of the found mycobiota, and to explore the ecological connection between fungi and the field history, including the potential of fungi in bioremediation and the production of novel bioactive compounds. Cysts were isolated from soil samples with a Spears apparatus and collected on a 150-µm sieve. The cysts were placed on potato dextrose agar, and maintained for two weeks at 27°C. Following fungal isolation and colony growing, the fungal DNA was extracted, the ITS region was amplified, and PCR products were sequenced. The study showed that the isolated fungal species belong to diverse phyla, including Ascomycota, Basidiomycota, and Mucoromycota. Ascomycota is represented by the families Clavicipitaceae, Sarocladiaceae, Nectriaceae, and Phaeosphaeriaceae. Basidiomycota is represented by the families Cerrenaceae, Polyporaceae, Phanerochaetaceae, and Meruliaceae, and the order Cantharellales. The family Mortierellaceae represents Mucoromycota. The members of Ascomycota and Basidiomycota both depict the field history. Ascomycota indicate the fungal infection is of recent origin, while Basidiomycota point toward the preceding host plants, enabling the plant field colonization history to be traced chronologically.

Hydrocarbon Degradation and Enzyme Activities of Aspergillus oryzae and Mucor irregularis Isolated from Nigerian Crude Oil-Polluted Sites.

Many free-living saprobic fungi are nature recruited organisms for the degradation of wastes, ranging from lignocellulose biomass to organic/inorganic chemicals, aided by their production of enzymes. In this study, fungal strains were isolated from contaminated crude-oil fields in Nigeria. The dominant fungi were selected from each site and identified as Aspergillus oryzae and Mucor irregularis based on morphological and molecular characterization, with site percentage incidences of 56.67% and 66.70%, respectively. Selected strains response/tolerance to complex hydrocarbon (used engine oil) was studied by growing them on Bushnell Haas (BH) mineral agar supplemented with the hydrocarbon at different concentrations, i.e., 5%, 10%, 15%, and 20%, with a control having dextrose. Hydrocarbon degradation potentials of these fungi were confirmed in BH broth culture filtrates pre-supplemented with 1% engine oil after 15 days of incubation using GC/MS. In addition, the presence of putative enzymes, laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) was confirmed in culture filtrates using appropriate substrates. The analyzed fungi grew in hydrocarbon supplemented medium with no other carbon source and exhibited 39.40% and 45.85% dose inhibition response (DIR) respectively at 20% hydrocarbon concentration. An enzyme activity test revealed that these two fungi produced more Lac than MnP and LiP. It was also observed through the GC/MS analyses that while A. oryzae acted on all hydrocarbon components in the used engine oil, M. irregularis only degraded the long-chain hydrocarbons and BTEX. This study confirms that A. oryzae and M. irregularis have the potential to be exploited in the bio-treatment and removal of hydrocarbons from polluted soils.

Soil Microfungi of the Colombian Natural Regions.

Although Colombia was one of the first tropical countries where an effort was made to gather mycological flora, contributions to the taxonomy, diversity, and ecology of soil microfungi are still scarce. In this study, the diversity of soil microfungi was studied collecting data from literature according to the Colombian natural regions: Andean, Amazonian, Caribbean, Orinoquía, Pacific, and Insular. The majority of the records comes from the Andean region, the most accessible to research. The other regions have been much less studied, with the Insular one with no data at all. International literature reported, up to now, ca. 300 different species of soil microfungi belonging to 126 different genera and 6 phyla (Ascomycota, Basidiomycota Mucoromycota, Glomeromycota, Mortierellomycota, and Olpidiomycota). Vescicular-Arbuscular fungi were widely investigated with Acaulospora and Glomus, the most recorded genera with ca. 20 species each. Ascomycota was the most diverse phylum with Penicillium, Aspergillus, and Fusarium representing the majority. Mortierella is strongly present in Mortierellomycota, and in the panorama of all recorded fungi, too. The other phyla and genera were less recorded. It is, therefore, evident the need to continue studying the soil microfungi in Colombia to have a better understanding of soil functioning and its ecosystem services.

Extracellular Enzymes and Bioactive Compounds from Antarctic Terrestrial Fungi for Bioprospecting.

Antarctica, one of the harshest environments in the world, has been successfully colonized by extremophilic, psychrophilic, and psychrotolerant microorganisms, facing a range of extreme conditions. Fungi are the most diverse taxon in the Antarctic ecosystems, including soils. Genetic adaptation to this environment results in the synthesis of a range of metabolites, with different functional roles in relation to the biotic and abiotic environmental factors, some of which with new biological properties of potential biotechnological interest. An overview on the production of cold-adapted enzymes and other bioactive secondary metabolites from filamentous fungi and yeasts isolated from Antarctic soils is here provided and considerations on their ecological significance are reported. A great number of researches have been carried out to date, based on cultural approaches. More recently, metagenomics approaches are expected to increase our knowledge on metabolic potential of these organisms, leading to the characterization of unculturable taxa. The search on fungi in Antarctica deserves to be improved, since it may represent a useful strategy for finding new metabolic pathways and, consequently, new bioactive compounds.

A meta-barcoding analysis of soil mycobiota of the upper Andean Colombian agro-environment.

Colombia is a country for which one of the highest biodiversity rates is reported, and one of the first in the tropical areas where an effort was made to gather information on indigenous fungi. Nevertheless, mycological data are still scarce and discontinuous, above all on soil fungi. The present study wanted to contribute to unveil the large soil fungal biodiversity in the upper Andean Colombian agro-ecosystems. The studied area is located in the department of Boyacà, considered with a notable economical value, partly devoted to subsistence agriculture. More than 150 described species were revealed in this study, belonging to 5 phyla with Ascomycota representing the dominant taxon. Basidiomycota and Zygomycota are also well represented, dominated by species of the genus Sebacina and Mortierella respectively, mainly distributed in the semi-natural plots (woodland and grassland). Most of the species are reported as first records for Colombia. Some of them are particularly interesting for their conservation significance such as Geoglossum fallax, which is the dominant species in the unimproved grassland plot. The bootstrap-based clustering analysis showed a different distribution of the species in orchards and non-cultivated areas as a possible response of the fungal community to different use of soil in the agro-environment.

Phytochemical Study of the Ecuadorian Species Lepechinia mutica (Benth.) Epling and High Antifungal Activity of Carnosol against Pyricularia oryzae.

The plant Lepechinia mutica (Benth.) Epling (family Lamiaceae) is endemic to Ecuador. In the present study, we report some major non-volatile secondary metabolites from the leaves and the chemistry of the essential oil distilled from the flowers. The main identified compounds were carnosol, viridiflorol, ursolic acid, oleanolic acid, chrysothol, and 5-hydroxy-4′,7-dimethoxy flavone. Their structures were determined by X-ray diffraction and NMR and MS techniques. The essential oil showed a chemical composition similar to that distilled from the leaves, but with some qualitative and quantitative differences regarding several minor compounds. The main constituents (>4%) were: δ-3-carene (24.23%), eudesm-7(11)-en-4-ol (13.02%), thujopsan-2-α-ol (11.90%), β-pinene (7.96%), valerianol (5.19%), and co-eluting limonene and β-phellandrene (4.47%). The volatile fraction was also submitted to enantioselective analysis on a β-cyclodextrin column, obtaining the separation and identification of the enantiomers for α-thujene, β-pinene, sabinene, α-phellandrene, limonene and β-phellandrene. Furthermore, the anti-fungal activity of non-volatile secondary metabolites was tested in vitro, with carnosol resulting in being very active against the “blast disease” caused by the fungus Pyricularia oryzae.

Composition, Antifungal and Antiproliferative Activities of the Hydrodistilled Oils from Leaves and Flower Heads of Pterocephalus nestorianus Nábelek.

This article reports the first study of the chemical composition, and antifungal and antiproliferative properties of the volatile extracts obtained by hydrodistillation of the flower heads and leaves of the traditional Kurdish medicinal plant Pterocephalus nestorianus Nábelek, collected in the wild. A total of 55 constituents, 43 of the flower heads' oil (PFO) and 46 of the leaves' oil (PLO), respectively, were identified by GC/MS, constituting 99.68% and 99.04% of the two oils, respectively. The oils were obtained in 0.15% and 0.10% yields (w/w), respectively, on air-dried vegetable material. The prevalent constituents of the PFO were α-terpineol (2.41%), α-linalool (6.42%), 6,10,14-trimethylpentadecan-2-one (2.59%), myristic acid (24.65%), and lauric acid (50.44%), while the major components of PLO were (E)-hex-2-enal (2.26%), (E)-hex-2-en-1-ol (2.04), myristic acid (34.03%), and lauric acid (50.35%). The two oils showed significant inhibitory and fungicidal activities against the medically important fungi Candida albicans, Candida tropicalis, Microsporum canis, and Trichophyton mentagrophytes, with minimum inhibitory concentration ranging from 0.7 to 3.3 mg/ml and minimum fungicidal concentration varying from 1.4 to 6.6 mg/ml. The antiproliferative activity of the two oils was assayed against one normal and six human tumor cell lines. Both oils showed selective cytotoxic activity, with IC50 values ranging from 1.4 to 3.3 µg/ml.

Chemical Composition and Antifungal Activity of Essential Oils from Flowers, Leaves, Rhizomes, and Bulbs of the Wild Iraqi Kurdish Plant Iris persica.

Essential oils-obtained by hydrodistillation of different parts of Iris persica L. were investigated for the first time by GC-FID and GC-MS; moreover, their antifungal activities were determined: 34,. 32, 27, and 17 compounds were identified in the oils from air-dried flowers, leaves, rhizomes and fresh. bulbs, respectively, representing ≥ 98% each oil. The major constituents of the flower essential oil were phenylethanol (24.8%) and furfural (13.8%), which, as the main component, constituted also 39.0% and 22.2% of the leaf and rhizome volatile fractions, respectively. Phenylacetaldehyde (37.1%) was the main constituent of the bulb volatile fraction. In in vitro tests, moderate antifungal activity was detected for the oils against strains of the human pathogenic fungal species Gandida albicans, Microsporum canis, and Trichophyton mentagrophytes, the plant-fungal pathogen Pyricularia oryzae, and the fungal food contaminant Aspergillus carbonarius,. The highest activity was exhibited by the essential oils from leaves and flowers, suggesting that they could be considered natural antimicrobial agents.

Fungal biodiversity in the periglacial soil of Dosdè Glacier (Valtellina, Northern Italy).

Periglacial areas are one of the least studied habitats on Earth, especially in terms of their fungal communities. In this work, both molecular and culture-dependent methods have been used to analyse the microfungi in soils sampled on the front of the East Dosdè Glacier (Valtellina, Northern Italy). Although this survey revealed a community that was rich in fungal species, a distinct group of psychrophilic microfungi has not been detected. Most of the isolated microfungi were mesophiles, which are well adapted to the sensitive climatic changes that occur in this alpine environment. A discrepancy in the results that were obtained by means of the two diagnostic approaches suggests that the used molecular methods cannot entirely replace traditional culture-dependent methods, and vice versa.

Triterpenoid glycosides from Medicago sativa as antifungal agents against Pyricularia oryzae.

The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.) tops and roots, the corresponding mixtures of prosapogenins from tops, and purified saponins and sapogenins against the causal agent of rice blast Pyricularia oryzae isolates are presented. In vitro experiments highlighted a range of activities, depending upon the assayed metabolite. The antifungal effects of the most promising prosapogenin mixture from alfalfa tops were confirmed by means of in planta tests using three different Italian cultivars of rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low blast resistance. The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure-activity relationship. Results indicate that prosapogenins are active compounds to prevent the fungal attack of P. oryzae on different rice cultivars. Therefore, if properly formulated, these substances could represent a promising and environmentally friendly treatment to control rice blast.

Lactarane sesquiterpenes from the European mushrooms Lactarius aurantiacus, L. subdulcis, and Russula sanguinaria.

The results of the first investigation of secondary metabolites occurring in intact and damaged fruiting bodies of the European mushrooms Lactarius aurantiacus, L. subdulcis, and Russula sanguinaria are reported. The pattern of sesquiterpenes in injured R. sanguinaria is dramatically different from that of Lactarius. The structure of the new furanolactarane 4, isolated from L. aurantiacus, was established on the basis of spectral data.

In vitro evaluation of nematophagous activity of fungal isolates.

Four filamentous fungi associated with nematodes were isolated and identified from litter samples collected in the Integral Natural Reserve "Bosco Siro Negri" (PV, Italy): Arthrobotrys dactyloides, Arthrobotrys oligospora var. oligospora, Pochonia bulbillosa, and Pochonia chlamydosporia var. catenulata. Their capacity to break down the nematode population was evaluated in vitro by means of simple and reproducible multiwell plates method. All fungal strains were able to cause a death-rate significantly different from the controls (p < 0.05). Precisely, A. dactyloides caused, on average, a 26% death rate increase in the nematode population compared to the control, A. oligospora var. oligospora 25%, P. bulbillosa 12%, and P. chlamidosporia var. catenulata 17%. The method has also allowed to determine the more active fungi as regards the prey's life cycle stage. The most active strains against nematodes (adults) were A. dactyloides and A. oligospora var. oligospora, known to attack adults or larval stages by means of tridimensional traps. On the contrary P. bulbillosa and P. chlamydosporia, known to attack mainly the nematode life stage of cysts, showed lower activity against adult nematodes.