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.

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea.

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillus protuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 µg/mL.

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.

The culturable mycobiota of a Mediterranean marine site after an oil spill: isolation, identification and potential application in bioremediation.

Bioremediation of marine environment could be the response to oil spills threats. In the present study the fungal community from a Mediterranean marine site chronically interested by oil spills was investigated. Sixty-seven taxa were isolated from water sample and 17 from sediments; for many of the identified species is the first report in seawater and sediments, respectively. The growth of 25% of the fungal isolates was stimulated by crude oil as sole carbon source. Four strains were selected to screen hydrocarbons degradation using the 2,6-dichlorophenol indophenol (DCPIP) colorimetric assay. A. terreus MUT 271, T. harzianum MUT 290 and P. citreonigrum MUT 267 displayed a high decolorization percentage (DP≥68%). A. terreus displayed also the highest decreases of hydrocarbons compounds (up to 40%) quantified by gas-chromatography analysis. These results suggest that the selected fungi could represent potential bioremediation agents with strong crude oil degradative capabilities.

Marine fungi as source of new hydrophobins.

Hydrophobins have been described as the most powerful surface-active proteins known. They are produced by filamentous fungi and exhibit a distinct amphiphilic structure determining their self-assembly at hydrophilic-hydrophobic interfaces and surfactant properties which have been demonstrated to be useful for several biotechnological applications. The marine environment represents a vast natural resource of new molecules produced by organisms growing in various stressful conditions. This study was focused on the screening of 100 marine fungi from Mycoteca Universitatis Taurinensis (MUT) for the identification of new hydrophobins. Four different methods were set up to extract hydrophobins of class I and II, from the mycelium or the culture broth of fungi. Six fungi were selected as the best producers of hydrophobins endowed with different characteristics. Their ability to form stable amphiphilic films and their emulsification capacity in the presence of olive oil was evaluated.

The antimicrobial potential of algicolous marine fungi for counteracting multidrug-resistant bacteria: phylogenetic diversity and chemical profiling.

Marine fungi represent an important but still largely unexplored source of novel and potentially bioactive secondary metabolites. The antimicrobial activity of nine sterile mycelia isolated from the green alga Flabellia petiolata collected from the Mediterranean Sea was tested on four antibiotic-resistant bacterial strains using extracellular and intracellular extracts obtained from each fungal strain. The isolated fungi were identified at the molecular level and assigned to one of the Dothideomycetes, Sordariomycetes or Eurotiomycetes classes. Following assessment of inhibition of bacterial growth (IC50), all crude extracts were subjected to preliminary (1)H NMR and TLC analysis. According to preliminary pharmacologic and spectroscopic/chromatographic results, extracts of fungal strains MUT 4865, classified as Beauveria bassiana, and MUT 4861, classified as Microascacea sp.2, were selected for LC-HRMS analysis. Chemical profiling of antibacterial extracts from MUT 4861 and MUT 4865 by LC HRMS allowed identification of the main components of the crude extracts. Several sphingosine bases were identified, including a compound previously unreported from natural sources, which gave a rationale to the broad spectrum of antibacterial activity exhibited.

Dothideomycetes and Leotiomycetes sterile mycelia isolated from the Italian seagrass Posidonia oceanica based on rDNA data.

Marine fungi represent a group of organisms extremely important from an ecological and biotechnological point of view, but often still neglected. In this work, an in-depth analysis on the systematic and the phylogenetic position of 21 sterile mycelia, isolated from Posidonia oceanica, was performed. The molecular (ITS and LSU sequences) analysis showed that several of them are putative new species belonging to three orders in the Ascomycota phylum: Pleosporales, Capnodiales and Helotiales. Phylogenetic analyses were performed using Bayesian Inference and Maximum Likelihood approaches. Seven sterile mycelia belong to the genera firstly reported from marine environments. The bioinformatic analysis allowed to identify five sterile mycelia at species level and nine at genus level. Some of the analyzed sterile mycelia could belong to new lineages of marine fungi.

Diversity, ecological role and potential biotechnological applications of marine fungi associated to the seagrass Posidonia oceanica.

The marine environment is characterized by high salinity and exerts a strong selective pressure on the biota, favouring the development of halo-tolerant microorganisms. Part of this microbial diversity is made up of fungi, important organisms from ecological and biotechnological points of view. In this study, for the first time, the qualitative and quantitative composition of the mycoflora associated to leaves, rhizomes, roots and matte of the seagrass Posidonia oceanica was estimated. A total of 88 fungal taxa, mainly belonging to Ascomycota, were identified by morphological and molecular methods. The most represented genera were Penicillium, Cladosporium and Acremonium. Most of the species (70) were selectively associated with one district; only two species (Penicillium chrysogenum var. chrysogenum and P. janczewskii) were isolated from all the districts. Moreover the capability to produce laccases, peroxidases and tannases by 107 fungal isolated by the different districts of P. oceanica was carried out. These results show that the mycoflora associated to P. oceanica is very rich and characterized by fungi able to produce ligninolytic enzymes and tannases useful to degrade and detoxify lignocellulose residues in presence of high salt concentrations. These fungi, hence, may play important ecological roles in marine environments but can also be very useful in different biotechnological areas.

Restriction fragment length polymorphism of the 5S-rRNA-NTS region: a rapid and precise method for plant identification.

Molecular genetic methods have several advantages over classical morphological and chemical analyses. The genetic method requires genotype instead than phenotype, therefore PCR-based techniques have been widely used for a rapid identification of plant species, varieties and chemotypes. Recently, the molecular discrimination of some higher plant species has been evaluated using sequences of a 5S-rRNA gene spacer region. The variation in the nontranscribed sequence (NTS) region has been used in a number of plant species for studying intraspecific variation, genome evolution, and phylogenetic reconstruction. Here, we describe a rapid method based on the use of the 5S-rRNA-NTS region as a tool for plant DNA fingerprinting, which combines PCR, sequencing and restriction fragment length polymorphism analyses.

HS-SPME-GC×GC-qMS volatile metabolite profiling of Chrysolina herbacea frass and Mentha spp. leaves.

Headspace solid-phase microextraction (HS-SPME) comprehensive two-dimensional (2D) gas chromatography combined with quadrupole-mass spectrometry (GC×GC-qMS) with dedicated comparative data elaboration was applied to separate chemical patterns arising from the interaction between some Mentha species and the herbivore Chrysolina herbacea, also known as the mint bug. Upon feeding on different Mentha species (Mentha spicata L., Mentha × piperita L. and Mentha longifolia L.), C. herbacea produced frass (faeces) which were characterized by a typical volatile fraction. HS-SPME GC×GC-qMS analysis of the complex volatile fraction of both mint leaf and C. herbacea frass was submitted to advanced fingerprinting analysis of 2D chromatographic data. 1,8-Cineole, found in the leaves of all the Mentha species examined, was oxidized, and C. herbacea frass yielded high rates of several hydroxy-1,8-cineoles, including 2α-hydroxy-, 3α-hydroxy-, 3ß-hydroxy- and 9-hydroxy-1,8-cineole. Upon insect feeding, several unknown oxidized monoterpenes, a p-menthane diol and three unknown phenylpropanoids were also detected in the frass volatiles. In M. longifolia, the occurrence of the monoterpene piperitenone oxide was found to be toxic and associated with insect death. The results of this work show that high throughput techniques such as HS-SPME and GC×GC-qMS fingerprint analysis are ideal tools to analyze complex volatile matrices, and provide a sensitive method for the direct comparison and chemical visualization of plant and insect emitted volatile components.

Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L.

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.

Comparative characterization of Santolina insularis chemotypes by essential oil composition, 5S-rRNA-NTS sequencing and EcoRV RFLP-PCR.

Santolina insularis (Genn ex Fiori) Arrig. is a medicinal plant whose essential oil shows antiviral and antibacterial activities and potent and selective cytotoxic activity against the human colon carcinoma cell line. The occurrence of several chemotypes makes the taxonomic identification of S. insularis hard to achieve. GC-MS essential oil analyses of four chemotypes (SI1, SI2, SI3 and SI4) revealed the presence of different percentages of santolina triene, beta-pinene, myrcene, beta-phellandrene, artemisia ketone and cis-chrysanthemol, allowing a chemical discrimination. Single fragments of the 5S-rRNA-NTS region of approximately 150, 170, 260 and 280bp were produced by SI1, SI2, SI3 and SI4, respectively, and the sequence alignment of the 5S-rRNA spacer region flanked by the 3'-and 5'-ends of the coding region confirmed a consistent difference between chemotypes. Furthermore, a PCR-RFLP method was applied. From the identified sequences, an EcoRV site could be found in chemotypes SI1, SI2 and SI3 in the 5S-rRNA spacer regions at 81 bp position; however, this site was absent in the chemotype SI4. This study, by showing remarkable chemical variation in the terpenoid profile and consistent genomic difference in the 5S-rRNA spacer regions, identified four chemotypes of S. insularis which could be grouped into two ecotypes, based on chemical and genomic analyses. The identification of specific gene sequences of the 5S-rRNA-NTS region and of a EcoRV site identified in this work can be used for a rapid and precise identification of the plant chemo-/ecotypes, complementing the essential oil chemical analysis.

Chemical and biomolecular characterization of Artemisia umbelliformis Lam., an important ingredient of the alpine liqueur "Genepi".

Artemisia umbelliformis Lam., an important alpine plant used for the preparation of flavored beverages, showed a remarkable intraspecific variability, at both genomic and gene product (secondary metabolites) levels. The variability of A. umbelliformis Lam. currently cultivated in Piedmont (Italy, Au1) and in Switzerland (Au2) was investigated by combining the chemical analysis of essential oil and sesquiterpene lactones and the molecular characterization of the 5S-rRNA-NTS gene by PCR and PCR-RFLP. Marked differences were observed between the two plants. Au1 essential oil contained alpha- and beta-thujones as the main components, whereas Au2 contained 1,8-cineole, borneol, and beta-pinene. Au1 sesquiterpene lactone fractions contained cis-8-eudesmanolide derivatives and Au2 the trans-6-germacranolide costunolide. Specific A. umbelliformis Au1 and Au2 primers were designed on the sequence of the 5S-rRNA gene spacer region. Furthermore, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was applied using RsaI and TaqI restriction enzymes. Chemical and biomolecular data contributed to the characterization of A. umbeliformis chemotypes.