Giorgio Gnavi

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.

Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi

The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841.

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 plants response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insects 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.

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

AbstractHeadspace 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-, 3β-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. FigureBecause of feeding, Chrysolina. herbacea transformed terpenoids in M. spicata and M.x piperita and not in M. longifolia. Chrysolina herbacea frass, after feeding of the two species, contained oxidized 1,8-cineole derivatives namely 2α-hydroxy-, 3α-hydroxy-, 3β-hydroxy- and 9-hydroxy-1,8-cineole.

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.

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.

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.

Chrysolina herbacea modulates jasmonic acid, cis-(+)-12-oxophytodienoic acid, (3R,7S)-jasmonoyl-l-isoleucine, and salicylic acid of local and systemic leaves in the host plant Mentha aquatica

Abstract Little information is available on the interaction between herbivorous insects and plants storing terpenoids. In this work we describe the response of the essential oil plant Mentha aquatica to the specialist herbivore Chrysolina herbacea. Feeding from C. herbacea induced a significant increase of jasmonic acid (JA) in both local and systemic M. aquatica leaves, whereas the content of the JA precursor, cis-(+)-12-oxophytodienoic acid (OPDA), was increased in local leaves and decreased in systemic leaves. The JA conjugate, (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile), was slightly increased in herbivore wounded (HW) local leaves, whereas its content significantly increased in systemic leaves. Herbivory by C. herbacea did not increase the content of salicylic acid (SA); however, SA showed a two-fold increase in HW systemic leaves. Our results indicate that also in plants producing direct defences, such as the essential oil plant M. aquatica, JA, and SA signalling is triggered by herbivory just like in plants that respond with indirect defence.

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.

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.