The genomes of Scedosporium between environmental challenges and opportunism.

Emerging fungal pathogens are a global challenge for humankind. Many efforts have been made to understand the mechanisms underlying pathogenicity in bacteria, and OMICs techniques are largely responsible for those advancements. By contrast, our limited understanding of opportunism and antifungal resistance is preventing us from identifying, limiting and interpreting the emergence of fungal pathogens. The genus Scedosporium (Microascaceae) includes fungi with high tolerance to environmental pollution, whilst some species can be considered major human pathogens, such as Scedosporium apiospermum and Scedosporium boydii. However, unlike other fungal pathogens, little is known about the genome evolution of these organisms. We sequenced two novel genomes of Scedosporium aurantiacum and Scedosporium minutisporum isolated from extreme, strongly anthropized environments. We compared all the available Scedosporium and Microascaceae genomes, that we systematically annotated and characterized ex novo in most cases. The genomes in this family were integrated in a Phylum-level comparison to infer the presence of putative, shared genomic traits in filamentous ascomycetes with pathogenic potential. The analysis included the genomes of 100 environmental and clinical fungi, revealing poor evolutionary convergence of putative pathogenicity traits. By contrast, several features in Microascaceae and Scedosporium were detected that might have a dual role in responding to environmental challenges and allowing colonization of the human body, including chitin, melanin and other cell wall related genes, proteases, glutaredoxins and magnesium transporters. We found these gene families to be impacted by expansions, orthologous transposon insertions, and point mutations. With RNA-seq, we demonstrated that most of these anciently impacted genomic features responded to the stress imposed by an antifungal compound (voriconazole) in the two environmental strains S. aurantiacum MUT6114 and S. minutisporum MUT6113. Therefore, the present genomics and transcriptomics investigation stands on the edge between stress resistance and pathogenic potential, to elucidate whether fungi were pre-adapted to infect humans. We highlight the strengths and limitations of genomics applied to opportunistic human pathogens, the multifactoriality of pathogenicity and resistance to drugs, and suggest a scenario where pressures other than anthropic contributed to forge filamentous human pathogens.

A Genomic and Transcriptomic Study on the DDT-Resistant Trichoderma hamatum FBL 587: First Genetic Data into Mycoremediation Strategies for DDT-Polluted Sites.

Trichoderma hamatum FBL 587 isolated from DDT-contaminated agricultural soils stands out as a remarkable strain with DDT-resistance and the ability to enhance DDT degradation process in soil. Here, whole genome sequencing and RNA-Seq studies for T. hamatum FBL 587 under exposure to DDT were performed. In the 38.9 Mb-genome of T. hamatum FBL 587, 10,944 protein-coding genes were predicted and annotated, including those of relevance to mycoremediation such as production of secondary metabolites and siderophores. The genome-scale transcriptional responses of T. hamatum FBL 587 to DDT exposure showed 1706 upregulated genes, some of which were putatively involved in the cellular translocation and degradation of DDT. With regards to DDT removal capacity, it was found upregulation of metabolizing enzymes such as P450s, and potentially of downstream DDT-transforming enzymes such as epoxide hydrolases, FAD-dependent monooxygenases, glycosyl- and glutathione-transferases. Based on transcriptional responses, the DDT degradation pathway could include transmembrane transporters of DDT, antioxidant enzymes for oxidative stress due to DDT exposure, as well as lipases and biosurfactants for the enhanced solubility of DDT. Our study provides the first genomic and transcriptomic data on T. hamatum FBL 587 under exposure to DDT, which are a base for a better understanding of mycoremediation strategies for DDT-polluted sites.

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.

Victoriomyces antarcticus gen. nov., sp. nov., a distinct evolutionary lineage of the Cephalothecaceae (Ascomycota) based on sequence-based phylogeny and morphology.

In this study, we propose a new genus, Victoriomyces, with a new species, Victoriomyces antarcticus, isolated from soil samples collected in Victoria Land, Antarctica. To determine its taxonomic status and evolutionary relationships, phylogenetic analysis was performed on DNA sequences from the nuclear 18S rRNA, 28S rRNA and the second largest subunit of RNA polymerase II (RPB2) genes. Victoriomyces antarcticus constitutes one well-supported distinct lineage within the Cephalothecaceae (family incertae sedis in Sordariomycetes), in which the only recognised asexual morphs belong to the genus Phialemonium and to Acremonium thermophilum. Victoriomyces antarcticus can be clearly distinguished from these taxa by means of DNA sequence analysis and its morphological traits that consist in having a Metarhizium-like asexual morph, dark red-coloured disk-like structures, immature bodies and the production of an intense red pigment in the growth media. Finally, we inferred the divergence time of V. antarcticus and the Cephalothecaceae using Bayesian analysis and secondary calibration. The holotype of V. antarcticus is FBL 165. The ex-type strain has been deposited as MUT 3686T and CCF 6158T. An additional strain of the species is FBL 577. The MycoBank number is MB 823713 for the genus and MB 823714 for the species.

On the molecular and morphological evolution of continental and insular Cryptorchestia species, with an additional description of C.garbinii (Talitridae).

Semi-terrestrial talitrid amphipods of the genus Cryptorchestia (sensu Lowry and Fanini 2013) associated with freshwater-soaked leaf litter were known to occur in inland lakes of Turkey and at the shores of the Black Sea. Before 2013 they had been reported as Orchestiacavimana and later as Cryptorchestiacavimana. In our phylogenetic tree, inferred from a mitochondrial and nuclear gene dataset (cytochrome oxidase I (COI), and histone H3 (H3), respectively), we show that these Turkish populations belong to Cryptochestiagarbinii, a common and widespread continental species, which is closely related to C.cavimana (endemic to Cyprus) and C.ruffoi (endemic to Rhodes). For the Turkish and European populations of C.garbinii, we found low levels of both genetic differentiation and morphological variation, and an age-related size variability (increasing at each moult) of the small lobe in the male gnathopod I merus, the main taxonomically diagnostic character for Cryptorchestia. A mainland (C.garbinii) versus insular isolation and in situ speciation (C.cavimana, and C.ruffoi) in the two east Mediterranean islands of Cyprus and Rhodes is discussed in relation to terrestrial Cryptorchestia species endemic to North East Atlantic volcanic islands (Azores, Canary Islands, and Madeira). The incorporation of five Mediterranean and Atlantic Orchestia species in the Bayesian analysis of the two genes (COI, and H3) indicated that both genera Orchestia and Cryptorchestia are not monophyletic.

Cryptorchestia ruffoi sp. n. from the island of Rhodes (Greece), revealed by morphological and phylogenetic analysis (Crustacea, Amphipoda, Talitridae).

A new Cryptorchestia species, Cryptorchestia ruffoi Latella & Vonk, sp. n. from the island of Rhodes in south-eastern Greece, can be distinguished on the basis of morphological and phylogenetic data. Morphological analysis and DNA sequencing of mitochondrial and nuclear protein-coding genes indicated that this species is related to Cryptorchestia cavimana (Cyprus) and Cryptorchestia garbinii (Mediterranean regions, with a recent northward expansion). Results supported a genetic separation between the Cryptorchestia species of the east Mediterranean regions and those of the northeast Atlantic volcanic islands examined in this study (Cryptorchestia canariensis, Cryptorchestia gomeri, Cryptorchestia guancha, and Cryptorchestia stocki from the Canary islands, Cryptorchestia monticola from Madeira, and Cryptorchestia chevreuxi from the Azores). The Mediterranean and Atlantic Cryptorchestia species appear to be also morphologically distinct. Cryptorchestia ruffoisp. n., Cryptorchestia cavimana, Cryptorchestia garbinii, and Cryptorchestia kosswigi (Turkish coast) clearly have a small lobe on the male gnathopod 1 merus. This character was the main diagnostic difference between Cryptorchestia (sensu Lowry, 2013) and Orchestia. However, among the six northeast Atlantic island Cryptorchestia species only Cryptorchestia stocki has a small lobe on the merus of gnathopod 1. Reduction or loss of the lobe in the Atlantic Island species cannot be ruled out; however, molecular phylogenetic analysis leads us to presume that this lobe independently evolved between the east Mediterranean Cryptorchestia species and Cryptorchestia stocki from Gran Canaria.

Arsenic-contaminated freshwater: assessing arsenate and arsenite toxicity and low-dose genotoxicity in Gammarus elvirae (Crustacea; Amphipoda).

Arsenic (As) contamination of freshwater is largely due to geogenic processes, but As is also released into the environment because of improper anthropic activities. The European regulatory limits in drinking water are of 10 µg L-1 As. However, knowledge of the genotoxic effects induced by low doses of As in freshwater environments is still scanty. This study was designed to investigate arsenate (As(V)) and arsenite (As(III)) toxicity and low-dose genotoxicity in Gammarus elvirae, which has proved to be a useful organism for genotoxicity assays in freshwater. As(V) and As(III) toxicity was assessed on the basis of the median lethal concentration, LC(50), while estimates of DNA damage were based on the Comet assay. The G. elvirae LC (50-240 h) value we calculated was 1.55 mg L-1 for As(V) and 1.72 mg L-1 for As(III). Arsenic exposure (240 h) at 5, 10, and 50 µg L-1 of As in assays with either arsenate or arsenite-induced DNA damage in hemocytes of G. elvirae in a concentration-dependent manner. Our study provides a basis for future genotoxic research on exposure to freshwater that contains low levels of arsenic.

An integrated study on Gammarus elvirae (Crustacea, Amphipoda): perspectives for toxicology of arsenic-contaminated freshwater.

The Italian region Latium is characterized by extensive quaternary volcanic systems that contribute greatly to arsenic (As) contamination of freshwater, including drinking water supplies. However, knowledge of the possible toxic effects in these aquatic environments is, despite being highly relevant to public health, still limited. In this paper, we approach this issue using Gammarus elvirae, an amphipod species that inhabits rivers and streams in central Italy, including Latium. We explored the possibility of using G. elvirae in the toxicology of freshwater by addressing the most relevant issues. First, we tested the usefulness of hemocytes from G. elvirae in determining non-specific DNA damage by means of the Comet assay after exposure (24 h and 7 days) to different river water samples in Latium; second, we provided an interpretative overview of the usefulness of hepatopancreatic epithelial cells of G. elvirae as a means of assessing toxicity after long-term exposure to As and other pollutants; third, the LC (50-240 h) value for G. elvirae was estimated for arsenate, which is usually the dominant arsenic species in surface waters. Our study sheds light on G. elvirae at different levels, providing a background for future toxicological research of freshwater.

A molecular and bioinformatic study on the ochratoxin A (OTA)-producing Aspergillus affinis (section Circumdati).

Aspergillus affinis (section Circumdati) is a novel ochratoxin A (OTA)-producing species found in submerged riparian decomposing leaves. However, very little is known about its role on the breakdown of plant debris and its ability to degrade carbohydrate polymers. Moreover, its OTA biosynthetic pathway has not yet been explored. In the present paper, we investigated the gene encoding the extracellular alpha-amylase (amyAa) of A. affinis within the evolution of the Aspergillus lineages in relation to the possible use of this enzyme in starch processing. The novel amyAa, despite being related to branches of the Aspergillus species of the sections Terrei and Flavi, formed a distinct phylogenetic branch, which may be of outstanding importance from a biotechnological point of view. Moreover, we identified the polyketide synthase gene (pks) putatively required for the first step of OTA biosynthesis in A. affinis. This otapks was examined in relation to a limited number of orthologous genes available from Aspergillus species of the sections Circumdati and Nigri. Our study highlights the importance of otapks as target genes in the treatment of ochratoxigenic Aspergillus species on a more comprehensive evolutionary basis.

A molecular study on bacterial resistance to arsenic-toxicity in surface and underground waters of Latium (Italy).

Latium, a region in central Italy, is known for its extensive volcanic areas that make a significant contribution to the arsenic (As) contamination of freshwater environments, even though some degree of As water pollution may be caused by human activities. The information available on indigenous As-resistant prokaryotes in aquatic environments of Latium is, however, still limited. In this study, we describe new bacteria that are resistant to arsenic toxicity and were isolated from the surface waters of Lake Vico and the Sacco River, two groundwater systems in Latium, as well as from bottled natural mineral water from the same region. The 16S rRNA gene sequence analysis for the As-resistant strains in lake and river waters points to a prevalence of ß- and γ-Proteobacteria, while α-Proteobacteria, Firmicutes and Bacteroidetes are represented to a lesser extent. By contrast, solely γ-Proteobacteria were isolated from groundwater samples. The presence of Actinobacteria was documented exclusively in bottled mineral water. In addition, we conducted a DNA sequence-based study on the gene codifying arsB, an As(III) efflux membrane protein pump related to arsenic resistance, for all the As-resistant bacterial isolates. A phylogenetic analysis was carried out on the newly sequenced 16S rRNA genes and arsB in the present study as well as on an additional 16S rRNA/arsB dataset we obtained previously from Lake Albano, from the Tiber and from a well in Bassano Romano located in Latium (Davolos and Pietrangeli, 2011). Overall, the phylogenetic diversity of As-resistant bacteria in underground water was very limited if compared with lentic and lotic waters. Lastly, our molecular data support the hypothesis that the horizontal gene transfer of ars in As-containing freshwater environments is not limited to closely-related genomes, but also occurs between bacteria that are distant from an evolutionary viewpoint, thereby indicating that such genetic events may be considered a source of microbial resistance to arsenic-toxicity.

Penicillium simile sp. nov. revealed by morphological and phylogenetic analysis.

The morphology of three phenetically identical Penicillium isolates, collected from the bioaerosol in a restoration laboratory in Italy, displayed macro- and microscopic characteristics that were similar though not completely ascribable to Penicillium raistrickii. For this reason, a phylogenetic approach based on DNA sequencing analysis was performed to establish both the taxonomic status and the evolutionary relationships of these three peculiar isolates in relation to previously described species of the genus Penicillium. We used four nuclear loci (both rRNA and protein coding genes) that have previously proved useful for the molecular investigation of taxa belonging to the genus Penicillium at various evolutionary levels. The internal transcribed spacer region (ITS1-5.8S-ITS2), domains D1 and D2 of the 28S rDNA, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and sequenced. Analysis of the rRNA genes and of the benA and cmd sequence data indicates the presence of three isogenic isolates belonging to a genetically distinct species of the genus Penicillium, here described and named Penicillium simile sp. nov. (ATCC MYA-4591(T)  = CBS 129191(T)). This novel species is phylogenetically different from P. raistrickii and other related species of the genus Penicillium (e.g. Penicillium scabrosum), from which it can be distinguished on the basis of morphological trait analysis.

Aspergillus affinis sp. nov., a novel ochratoxin A-producing Aspergillus species (section Circumdati) isolated from decomposing leaves.

Two ochratoxin A (OTA)-producing Aspergillus isolates, recently collected from submerged riparian decomposing leaves in Italy, were found to have a similar morphology to Aspergillus cretensis (subgenus Circumdati, section Circumdati). However, marked differences emerged between these two novel isolates and A. cretensis as the former displayed different colony features and had larger vesicles, metulae, phialides and conidia, as well as a distinct sclerotial form and size. In order to determine the taxonomic status and to infer the evolutionary relationships of these two morphologically identical isolates, a molecular phylogenetic analysis was performed on all the officially recognized lineages in the section Circumdati. The DNA sequences and the deduced amino acid residues from the nuclear loci were analysed. Both rRNA and protein coding genes were assessed, which are widely used to differentiate taxa belonging to genus Aspergillus at various evolutionary levels. The 5.8S rDNA gene and internal transcribed spacers (ITS), the D1/D2 domains of the 28S rDNA gene, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and then sequenced. The analysis of the rRNA regions and of the benA and cmd sequence data indicated that the two isogenic isolates belonged to a genetically distinct OTA-producing species of the genus Aspergillus. The isolates are proposed as representing a novel species, Aspergillus affinis sp. nov., with the type strain ATCC MYA-4773T=CBS 129190=417). Phylogenetically, A. affinis sp. nov. appeared to be very closely related to A. cretensis, from which it could be distinguished by means of a morphological trait analysis.

A molecular phylogenetic study of the freshwater talitrid amphipod Orchestia cavimana (Crustacea).

In this paper we performed a molecular phylogenetic study of Orchestia cavimana, the sole talitrid amphipod inhabiting beaches of European freshwater lakes and rivers. For that purpose, we have PCR amplified and sequenced regions of the mitochondrial cytochrome oxidase subunit I (COI) gene, basing our analysis on both nucleotide and amino acid sequences and considering also structural classes of the COI enzyme. Phylogenetic analyses were conducted by neighbour-joining (NJ) and maximum-parsimony (MP) methods comparing homologous sequences of talitrids and other Crustacea. In both NJ and MP trees, O. cavimana shows a basal placement with respect to other talitrid amphipods.