Knufia obscura sp. nov. and Knufia victoriae sp. nov., two new species from extreme environments.

Six strains of black meristematic fungi were isolated from Antarctic soils, gasoline car tanks and from the marine alga Flabellia petiolata. These fungi were characterized by morphological, physiological and phylogenetic analyses. According to the maximum-likelihood analysis reconstructed with ITS and LSU sequences, these strains belonged to the genus Knufia. Knufia obscura sp. nov. (holotype CBS 148926) and Knufia victoriae sp. nov. (holotype CBS 149015) are proposed as two novel species and descriptions of their morphological, physiological and phylogenetic features are presented. Based on the maximum-likelihood analyses, K. obscura was closely related to Knufia hypolithi (99 % bootstrap support), while K. victoriae clustered in the clade of Knufia cryptophialidica and Knufia perfecta (93 % bootstrap support). Knufia victoriae, recorded in Antarctic soil samples, had a psychrophilic behaviour, with optimal growth between 10 and 15 °C and no growth recorded at 20 °C. Knufia obscura, from a gasoline car tank and algae, displayed optimal growth between 20 and 25 °C and was more tolerant to salinity than K. victoriae.

Marine Fungi from the Sponge Grantia compressa: Biodiversity, Chemodiversity, and Biotechnological Potential.

The emergence of antibiotic resistance and viruses with high epidemic potential made unexplored marine environments an appealing target source for new metabolites. Marine fungi represent one of the most suitable sources for the discovery of new compounds. Thus, the aim of this work was (i) to isolate and identify fungi associated with the Atlantic sponge Grantia compressa; (ii) to study the fungal metabolites by applying the OSMAC approach (one strain; many compounds); (iii) to test fungal compounds for their antimicrobial activities. Twenty-one fungal strains (17 taxa) were isolated from G. compressa. The OSMAC approach revealed an astonishing metabolic diversity in the marine fungus Eurotium chevalieri MUT 2316, from which 10 compounds were extracted, isolated, and characterized. All metabolites were tested against viruses and bacteria (reference and multidrug-resistant strains). Dihydroauroglaucin completely inhibited the replication of influenza A virus; as for herpes simplex virus 1, total inhibition of replication was observed for both physcion and neoechinulin D. Six out of 10 compounds were active against Gram-positive bacteria with isodihydroauroglaucin being the most promising compound (minimal inhibitory concentration (MIC) 4-64 µg/mL) with bactericidal activity. Overall, G. compressa proved to be an outstanding source of fungal diversity. Marine fungi were capable of producing different metabolites; in particular, the compounds isolated from E. chevalieri showed promising bioactivity against well-known and emerging pathogens.

Wastewater-Agar as a selection environment: A first step towards a fungal in-situ bioaugmentation strategy.

Viable and metabolically active fungi in toxic mixed liquors, treating landfill leachates and municipal wastewaters, were identified by culture depending methods. A selective culture medium consisting of wastewater and agar (WA) restrained fungi that could be randomly present (94% of the 51 taxa retrieved on WA were sample-specific), overcoming the problem of fast growing fungi or mycoparasite fungi. Moreover, WA allowed the isolation of fungi with a possible role in the degradation of pollutants typically present in the two wastewaters. Phoma medicaginis var. medicaginis, Chaetomium globosum, and Geotrichum candidum were mainly found in municipal wastewater, whereas Pseudallescheria boydii, Scedosporium apiospermum, Aspergillus pseudodeflectus, and Scopulariopsis brevicaulis were typical of landfill leachate.

Control and prevention measures for legionellosis in hospitals: A cross-sectional survey in Italy.

Risk assessment, environmental monitoring, and the disinfection of water systems are the key elements in preventing legionellosis risk. The Italian Study Group of Hospital Hygiene of the Italian Society of Hygiene, Preventive Medicine, and Public Health and the Italian Multidisciplinary Society for the Prevention of Health Care-Associated Infections carried out a national cross-sectional survey to investigate the measures taken to prevent and control legionellosis in Italian hospitals. A multiple-choice questionnaire was developed, comprising 71 questions regarding hospital location, general characteristics, clinical and environmental surveillance, and control and preventive measures for legionellosis in 2015. Overall, 739 hospitals were enrolled from February to June 2017, and 178 anonymous questionnaires were correctly completed and evaluated (response rate: 24.1%). The survey was conducted using the SurveyMonkey® platform, and the data were analyzed using Stata 12 software. Of the participating hospitals, 63.2% reported at least one case of legionellosis, of which 28.2% were of proven nosocomial origin. The highest case numbers were reported in the Northern Italy, in hospitals with a pavilion structure or cooling towers, and in hospitals with higher numbers of beds, wards and operating theaters. Laboratory diagnosis was performed using urinary antigen testing alone (31.9%), both urinary antigen testing and single antibody titer (17.8%), or with seroconversion also added (21.5%). Culture-based or molecular investigations were performed in 28.8% and 22.1% of the clinical specimens, respectively. The water systems were routinely tested for Legionella in 97.4% of the hospitals, 62% of which detected a positive result (> 1000 cfu/L). Legionella pneumophila serogroup 2-15 was the most frequently isolated species (58.4%). The most common control measures were the disinfection of the water system (73.7%), mostly through thermal shock (37.4%) and chlorine dioxide (34.4%), and the replacement (69.7%) or cleaning (70.4%) of faucets and showerheads. A dedicated multidisciplinary team was present in 52.8% of the hospitals, and 73% of the hospitals performed risk assessment. Targeted training courses were organized in 36.5% of the hospitals, involving nurses (30.7%), physicians (28.8%), biologists (21.5%), technicians (26.4%), and cleaners (11%). Control and prevention measures for legionellosis are present in Italian hospitals, but some critical aspects should be improved. More appropriate risk assessment is necessary, especially in large facilities with a high number of hospitalizations. Moreover, more sensitive diagnostic tests should be used, and dedicated training courses should be implemented.

Fungi from industrial tannins: potential application in biotransformation and bioremediation of tannery wastewaters.

Tannins are a complex family of polyphenolic compounds, widely distributed in the plant kingdom where they act as growth inhibitors towards many microorganisms including bacteria, yeasts, and fungi. Tannins are one of the major components of tannery wastewaters and may cause serious environmental pollution. In the present study, four different tannins (the hydrolysable chestnut ellagitannin and tara gallotannin and the condensed quebracho and wattle tannins) were characterized from a mycological point of view with the aim of selecting fungal strains capable of growing in the presence of high tannin concentration and thus potentially useful in industrial biotransformations of these compounds or in the bioremediation of tannery wastewaters. A total of 125 isolates of filamentous fungi belonging to 10 species and four genera (Aspergillus, Paecilomyces, Penicillium, and Talaromyces) were isolated from the tannin industrial preparations. Miniaturized biotransformation tests were set up with 10 fungal strains and the high-performance liquid chromatography (HPLC) analysis pointed out a strong activity of all the tested fungi on both chestnut and tara tannins. Two strains (Aspergillus tubingensis MUT 990 and Paecilomyces variotii MUT 1125), tested against a real tannery wastewater, were particularly efficient in chemical oxygen demand (COD) and tannin removal (> 60%), with a detoxification above 74%. These results indicate that these fungi are potentially exploitable in the treatment of tannery wastewaters.

Tourism affects microbial assemblages in show caves.

Anthropogenic disturbance on natural ecosystems is growing in frequency and magnitude affecting all ecosystems components. Understanding the response of different types of biocoenosis to human disturbance is urgently needed and it can be achieved by adopting a metacommunity framework. With the aid of advanced molecular techniques, we investigated sediment communities of Fungi, Bacteria and Archaea in four Italian show caves, aiming to disentangle the effects induced by tourism on their diversity and to highlight changes in the driving forces that shape their community composition. We modelled diversity measures against proxies of tourism pressure. With this approach we demonstrate that the cave tourism has a direct effect on the community of Bacteria and an indirect influence on Fungi and Archaea. By analysing the main driving forces influencing the community composition of the three microbial groups, we highlighted that stochastic factors override dispersal-related processes and environmental selection in show caves compared to undisturbed areas. Thanks to this approach, we provide new perspectives on the dynamics of microbial communities under human disturbance suggesting that a proper understanding of the underlying selective mechanisms requires a comprehensive and multi-taxonomic approach.

Microbial diversity and proxy species for human impact in Italian karst caves.

To date, the highly adapted cave microbial communities are challenged by the expanding anthropization of these subterranean habitats. Although recent advances in characterizing show-caves microbiome composition and functionality, the anthropic effect on promoting the establishment, or reducing the presence of specific microbial guilds has never been studied in detail. This work aims to investigate the whole microbiome (Fungi, Algae, Bacteria and Archaea) of four Italian show-caves, displaying different environmental and geo-morphological conditions and one recently discovered natural cave to highlight potential human-induced microbial traits alterations. Results indicate how show-caves share common microbial traits in contrast to the natural one; the first are characterized by microorganisms related to outdoor environment and/or capable of exploiting extra inputs of organic matter eventually supplied by tourist flows (i.e. Chaetomium and Phoma for fungi and Pseudomonas for bacteria). Yet, variation in microalgae assemblage composition was reported in show-caves, probably related to the effect of the artificial lighting. This study provides insights into the potential microbiome cave contamination by human-related bacteria (e.g. Lactobacillus and Staphylococcus) and commensal/opportunistic human associated fungi (e.g. Candida) and dermatophytes. This work is critical to untangle caves microbiome towards management and conservation of these fragile ecosystems.

The Culturable Mycobiota of Sediments and Associated Microplastics: From a Harbor to a Marine Protected Area, a Comparative Study.

Fungi are an essential component of marine ecosystems, although little is known about their global distribution and underwater diversity, especially in sediments. Microplastics (MPs) are widespread contaminants worldwide and threaten the organisms present in the oceans. In this study, we investigated the fungal abundance and diversity in sediments, as well as the MPs, of three sites with different anthropogenic impacts in the Mediterranean Sea: the harbor of Livorno, the marine protected area "Secche della Meloria"; and an intermediate point, respectively. A total of 1526 isolates were cultured and identified using a polyphasic approach. For many of the fungal species this is the first record in a marine environment. A comparison with the mycobiota associated with the sediments and MPs underlined a "substrate specificity", highlighting the complexity of MP-associated fungal assemblages, potentially leading to altered microbial activities and hence changes in ecosystem functions. A further driving force that acts on the fungal communities associated with sediments and MPs is sampling sites with different anthropogenic impacts.

Insights on Lulworthiales Inhabiting the Mediterranean Sea and Description of Three Novel Species of the Genus Paralulworthia.

The order Lulworthiales, with its sole family Lulworthiaceae, consists of strictly marine genera found on a wide range of substrates such as seagrasses, seaweeds, and seafoam. Twenty-one unidentified Lulworthiales were isolated in previous surveys aimed at broadening our understanding of the biodiversity hosted in the Mediterranean Sea. Here, these organisms, mostly found in association with Posidonia oceanica and with submerged woods, were examined using thorough multi-locus phylogenetic analyses and morphological observations. Maximum-likelihood and Bayesian phylogeny based on nrITS, nrSSU, nrLSU, and four protein-coding genes led to the introduction of three novel species of the genus Paralulworthia: P. candida, P. elbensis, and P. mediterranea. Once again, the marine environment is a confirmed huge reservoir of novel fungal lineages with an under-investigated biotechnological potential waiting to be explored.

Low density polyethylene degradation by filamentous fungi.

Polyethylene (PE) is the most abundant non-degradable plastic waste, posing a constant and serious threat to the whole ecosystem. In the present study, the fungal community of plastic wastes contaminating a landfill soil has been studied. After 6 months of enrichment, 95 fungi were isolated, mostly belonging to the Ascomycota phylum. They were screened under in vitro condition: most of fungi (97%) were capable of growing in the presence of PE powder (5-10 g L-1) as sole carbon source. Fusarium strains better tolerated high concentration of PE. Up to 13 strains were chosen for further degradation trails, where the process was monitored by respirometry tests and by observing changes in PE chemical and physical structure by FTIR analysis and SEM images. Major results were observed for Fusarium oxysporum, Fusarium falciforme and Purpureocillum lilacinum, as they caused strong oxidation phenomena and changes in the PE film morphology. Results suggested that the initial oxidation mechanisms targeted first the methyl terminal groups. Changes in the infrared spectra were strongly strain-dependent, denoting the activation of different degradation pathways. Through the SEM analysis, the actual damages provoked by fungi were observed, including swellings, pits and furrows, bumps and partial exfoliations. Considering the rising concern about plastic disposal worldwide, the ability of these fungi to colonize PE and utilize it as carbon source is of great interest, as no pretreatments and pro-oxidant stimulants were needed.

Fungal Diversity in the Neptune Forest: Comparison of the Mycobiota of Posidonia oceanica, Flabellia petiolata, and Padina pavonica.

Fungi are widely distributed in the Oceans, interact with other organisms and play roles that range from pathogenic to mutualistic. The present work focuses on the characterization of the cultivable mycobiota associated with the seagrass Posidonia oceanica (L.) Delile collected off the Elba Island (Italy). We identified 102 taxa (mainly Ascomycota) by the mean of a polyphasic approach. Leaves, rhizomes, roots and matte were characterized by unique mycobiota revealing a "plant-part-specificity." The comparison with the mycobiota associated with the green alga Flabellia petiolata and the brown alga Padina pavonica underlined a "substrate specificity." Indeed, despite being part of the same phytocoenosis, these photosynthetic organisms recruit different fungal communities. The mycobiota seems to be necessary for the host's defense and protection, playing, in this way, remarkable ecological roles. Among the 61 species detected in association with P. oceanica (including two species belonging to the newly introduced genus Paralulworthia), 37 were reported for the first time from the Mediterranean Sea.

Genome Sequence of Trichoderma lixii MUT3171, A Promising Strain for Mycoremediation of PAH-Contaminated Sites.

Mono- and polycyclic aromatic hydrocarbons (PAHs) are widespread and recalcitrant pollutants that threaten both environmental and human health. By exploiting the powerful enzymatic machinery of fungi, mycoremediation in contaminated sites aims at removing a wide range of pollutants in a cost-efficient and environmentally friendly manner. Next-generation sequencing (NGS) techniques are powerful tools for understanding the molecular basis of biotransformation of PAHs by selected fungal strains, allowing genome mining to identify genetic features of biotechnological value. Trichoderma lixii MUT3171, isolated from a historically PAH-contaminated soil in Italy, can grow on phenanthrene, as a sole carbon source. Here, we report the draft genome sequence of T. lixii MUT3171 obtained with high-throughput sequencing method. The genome of T. lixii MUT3171 was compared with other 14 Trichoderma genomes, highlighting both shared and unique features that can shed a light on the biotransformation of PAHs. Moreover, the genes potentially involved in the production of important biosurfactants and bioactive molecules have been investigated. The gene repertoire of T. lixii MUT3171 indicates a high degrading potential and provides hints on putative survival strategies in a polluted environment.

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Elbamycellarosea gen. et sp. nov. (Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea.

Elbamycellarosea sp. nov., introduced in the new genus Elbamycella, was collected in the Mediterranean Sea in association with the seagrass Posidoniaoceanica and with the brown alga Padinapavonica. The affiliation of the new taxon to the family Juncigenaceae is supported by both morphology and phylogenetic inference based on a combined nrSSU and nrLSU sequence dataset. Maximum-likelihood and Bayesian phylogeny proved Elbamycella gen. nov. as a distinct genus within Juncigenaceae. The new genus has been compared with closely related genera and is characterised by a unique suite of characters, such as ascospores with polar appendages and peculiar shape and dimension of ascomata and asci.

The culturable mycobiota associated with the Mediterranean sponges Aplysina cavernicola, Crambe crambe and Phorbas tenacior.

Marine fungi are part of the huge and understudied biodiversity hosted in the sea. To broaden the knowledge on fungi inhabiting the Mediterranean Sea and their role in sponge holobiont, three sponges namely Aplysina cavernicola, Crambe crambe and Phorbas tenacior were collected in Villefranche sur Mer, (France) at about 25 m depth. The fungal communities associated with the sponges were isolated using different techniques to increase the numbers of fungi isolated. All fungi were identified to species level giving rise to 19, 13 and 3 species for P. tenacior, A. cavernicola and C. crambe, respectively. Of note, 35.7% and 50.0% of the species detected were either reported for the first time in the marine environment or in association with sponges. The mini-satellite analysis confirmed the uniqueness of the mycobiota of each sponge, leading to think that the sponge, with its metabolome, may shape the microbial community.

Molecular and Microbiological Insights on the Enrichment Procedures for the Isolation of Petroleum Degrading Bacteria and Fungi.

Autochthonous bioaugmentation, by exploiting the indigenous microorganisms of the contaminated environment to be treated, can represent a successful bioremediation strategy. In this perspective, we have assessed by molecular methods the evolution of bacterial and fungal communities during the selective enrichment on different pollutants of a soil strongly polluted by mixtures of aliphatic and polycyclic hydrocarbons. Three consecutive enrichments were carried out on soil samples from different soil depths (0-1, 1-2, 2-3 m), and analyzed at each step by means of high-throughput sequencing of bacterial and fungal amplicons biomarkers. At the end of the enrichments, bacterial and fungal contaminants degrading strains were isolated and identified in order to (i) compare the composition of enriched communities by culture-dependent and culture-independent molecular methods and to (ii) obtain a collection of hydrocarbon degrading microorganisms potentially exploitable for soil bioremediation. Molecular results highlighted that for both bacteria and fungi the pollutant had a partial shaping effect on the enriched communities, with paraffin creating distinct enriched bacterial community from oil, and polycyclic aromatic hydrocarbons generally overlapping; interestingly neither the soil depth or the enrichment step had significant effects on the composition of the final enriched communities. Molecular analyses well-agreed with culture-dependent analyses in terms of most abundant microbial genera. A total of 95 bacterial and 94 fungal strains were isolated after selective enrichment procedure on different pollutants. On the whole, isolated bacteria where manly ascribed to Pseudomonas genus followed by Sphingobacterium, Bacillus, Stenothrophomonas, Achromobacter, and Serratia. As for fungi, Fusarium was the most abundant genus followed by Trichoderma and Aspergillus. The species comprising more isolates, such as Pseudomonas putida, Achromobacter xylosoxidans and Ochromobactrum anthropi for bacteria, Fusarium oxysporum and Fusarium solani for fungi, were also the dominant OTUs assessed in Illumina.

Tannery mixed liquors from an ecotoxicological and mycological point of view: Risks vs potential biodegradation application.

Fungi are known to be present in the activated sludge of wastewater treatment plants (WWTP). Their study should be at the base of an overall vision of the plant effectiveness and of effluents sanitary impact. Moreover, it could be fundamental for the implementation of successful bioaugmentation strategies aimed at the removal of recalcitrant or toxic compounds. This is one of the first studies on the cultivable autochthonous mycoflora present in the mixed liquors of two WWTP treating either vegetable or chromium tannery effluents. All samples showed a risk associated with potential pathogens or toxigenic species and high ecotoxicity (Lepidium sativum and Raphidocelis subcapitata were the most sensitive organisms). Diverse fungal populations developed, depending on the origin of the samples (63% of the 102 identified taxa were sample-specific). The use of a fungistatic was determinant for the isolation and, thus, for the identification of sample-specific species with a lower growth rate. The incubation temperature also affected the mycoflora composition, even though at lower extent. A selective medium, consisting of agarised wastewater, allowed isolating fungi with a biodegradation potential. Pseudallescheria boydii/Scedosporium apiospermum species complex was ubiquitously dominant, indicating a possible role in the degradation of pollutants in both WWTP. Other species, i.e. Trichoderma spp., Trematosphaeria grisea, Geotrichum candidum, Lichtheimia corymbifera, Acremonium furcatum, Penicillium simplicissimum, Penicillium dangeardii, Fusarium solani, Scopulariopsis brevicaulis potentially could be involved in the degradation of specific pollutants of vegetable or chromium tannery wastewaters. However, several of these fungi are potential pathogens and their application, for an in situ treatment, must be carefully evaluated.

The culturable mycobiota of Flabellia petiolata: First survey of marine fungi associated to a Mediterranean green alga.

Algae-inhabiting marine fungi represent a taxonomically and ecologically interesting group of microorganisms still largely neglected, especially in temperate regions. The aim of this study was to isolate and to identify the culturable mycobiota associated with Flabellia petiolata, a green alga frequently retrieved in the Mediterranean basin. Twenty algal thalli were collected from two different sampling sites in the Mediterranean Sea (Elba Island, Italy). A polyphasic approach showed the presence of a relevant alga-associated mycobiota with 64 taxa identified. The fungal isolates belonged mainly to Ascomycota (61 taxa), while only three Basidiomycota were detected. The phylogenetic position of sterile mycelia and cryptic taxa, inferred on the basis of LSU partial region, highlighted the presence of putative new phylogenetic lineages within Dothideomycetes and Sordariomycetes. This work represents the first quali-quantitative analysis of the culturable mycobiota associated to a green alga in the Mediterranean Sea.

Old Yellow Enzyme homologues in Mucor circinelloides: expression profile and biotransformation.

The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, α-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.

Influence of plant genotype on the cultivable fungi associated to tomato rhizosphere and roots in different soils.

Rhizosphere and root-associated microbiota are crucial in determining plant health and in increasing productivity of agricultural crops. To date, research has mainly focused on the bacterial dimension of the microbiota. However, interest in the mycobiota is increasing, since fungi play a key role in soil ecosystems. We examined the effect of plant genotype, soil, and of Fusarium oxysporum f. sp. lycopersici (Fol) on the cultivable component of rhizosphere and root-associated mycobiota of tomato. Resistant and susceptible varieties were cultivated on two different soils (A and B), under glasshouse conditions. Isolated fungi were identified by morphological and molecular approaches. Differences were found between the rhizosphere and the roots, which in general displayed a lower number of species. The structure of the mycobiota was significantly affected by the soil type in the rhizosphere as well as by the plant genotype within the roots (NPERMANOVA, p < 0.05). The addition of Fol changed the community structure, particularly in soil A, where Penicillium spp. and Fusarium spp. were the dominant responding fungi. Overall, the results indicated that i) soil type and plant genotype affect the fungal communities; ii) plant roots select few species from the rhizosphere; and iii) the fungal community structure is influenced by Fol.