Nanopore Sequencing and Bioinformatics for Rapidly Identifying Cultural Heritage Spoilage Microorganisms

Microbiological methodologies allow understanding the causes that lead to the development of a certain microbial community colonizing an artistic surface, to characterize its composition and describe its role in the deterioration of the constituent materials. Metagenomics allows identifying microbial communities directly in their natural environments, bypassing the need for isolation and cultivation of individual species, thus providing a more comprehensive picture of the biodiversity present on a surface compared with standard cultivation methods. Furthermore, molecular analyses require small amounts of material, favoring the preservation of the artistic surface during sampling. Here, we verified the suitability of a protocol consisting in DNA extraction with micro-invasive sampling, using adhesive tape, PCR amplification with universal primers [bacteria (16S), fungi (ITS), and Viridiplantae (18S)], and amplicon sequencing by Oxford Nanopore Technologies (ONT) in the hypogeum of Basilica di San Nicola in Carcere Church (Rome, Italy). Sequence data were analyzed with a bioinformatic pipeline customized for pinpointing cultural heritage spoiling organisms, named "AmpLIcon SequencIng Analysis" (ALISIA). These data were integrated with traditional microbiology techniques that allowed the isolation of cultivable bacteria; three species were also characterized through their capability of biofilm formation and antibiotic resistance. Further, Fourier-transform infrared spectroscopy (FTIR) spectroscopy was performed to characterize the main products present on the masonry surface providing indications on the type of decay present. This novel biological workflow represents a powerful opportunity to investigate the microbial colonization of artistic surfaces aimed at implementing preservation strategies of cultural heritage from bio-spoilage

The Neolithic site “La Marmotta”. DNA metabarcoding to identify the microbial deterioration of waterlogged archeological wood

Introduction: The evaluation of biological degradation of waterlogged archeological wood is crucial to choose the conservative and protective treatments to be applied to the wooden material. The waterlogged environmental conditions are characterized by oxygen scarcity, only allowing the growth of adapted microbes capable to degrade the organic wooden material, mainly erosion bacteria and softrot fungi. In this work, we characterized and evaluated the biodegradation state and the microbial communities of wooden fragments preserved in storage tanks. These were preserved by waterlogging within the Neolithic village “La Marmotta,” currently found under the Bracciano Lake (Lazio, Italy). Methods: The waterlogged wood samples were first identified taxonomically with an optical microscope, also allowing an evaluation of their preservation state. The microbial community was then evaluated through the sequencing of Internal Transcribed Spacer sequences for fungi and 16S for bacteria with the Oxford Nanopore Technologies (ONT) MinION platform. Results: The identified microbial community appears to be consistent with the waterlogged samples, as many bacteria attributable to the erosion of wood and ligninolytic fungi have been sequenced. Discussion: The reported results highlight the first use of targeted metabarcoding by ONT applied to study the biodeterioration of waterlogged archeological wood

The effect of mushroom culture filtrates on the inhibition of mycotoxins produced by Aspergillus flavus and Aspergillus carbonarius

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B-1 (AFB(1)), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB(1) or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB(1) in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB(1) by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan(R), by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis

The Lipoxygenase Lox1 Is Involved in Light‐ and Injury-Response, Conidiation, and Volatile Organic Compound Biosynthesis in the Mycoparasitic Fungus Trichoderma atroviride

18 páginas, 10 figuras. -- The first publication by Frontiers Media is avalilable at https://www.frontiersin.org/articles/10.3389/fmicb.2020.02004/fullThe necrotrophic mycoparasite Trichoderma atroviride is a biological pest control agent frequently applied in agriculture for the protection of plants against fungal phytopathogens. One of the main secondary metabolites produced by this fungus is 6-pentyl-α-pyrone (6-PP). 6-PP is an organic compound with antifungal and plant growth-promoting activities, whose biosynthesis was previously proposed to involve a lipoxygenase (Lox). In this study, we investigated the role of the single lipoxygenase-encoding gene lox1 encoded in the T. atroviride genome by targeted gene deletion. We found that light inhibits 6-PP biosynthesis but lox1 is dispensable for 6-PP production as well as for the ability of T. atroviride to parasitize and antagonize host fungi. However, we found Lox1 to be involved in T. atroviride conidiation in darkness, in injury-response, in the production of several metabolites, including oxylipins and volatile organic compounds, as well as in the induction of systemic resistance against the plant-pathogenic fungus Botrytis cinerea in Arabidopsis thaliana plants. Our findings give novel insights into the roles of a fungal Ile-group lipoxygenase and expand the understanding of a light-dependent role of these enzymes.This research was supported by the Austrian Science Fund (FWF; grant P32179-B), Tyrolean Science Fund (TWF; grant number AP718021) and the doctoral program BioApp from the University of InnsbruckPeer reviewe

LIPID MEDIATED CROSS-KINGDOM COMMUNICATION BETWEEN HOSTS AND PATHOGENS

Which are the main biological functions of lipids? Lipids have a structural role in forming cell membranes and play a role in energy storage. Current knowledge indicates that the lipids have an additional role in cell signaling. When a fungal pathogen contacts the host, the cell surfaces can exchange signals, among which, several are lipids. Lipids occur in fungi not only as major constituents of the membrane system, but also as cell wall components, as storage material in abundant and readily observed lipid bodies, and in some cases, as extracellular products. Fungi contain a various set of lipids, including fatty acids, oxylipins, sphingolipids, phospholipids, glycolipids and sterols. We are going to examine some fungal lipid classes involved in the pathogenic interaction with the host. These signals can confer different information. In some cases can trigger host immune responses; conversely, they may function as virulence factors altering the normal homeostasis of the host or even causing the death

The Role of Aflatoxins in <em>Aspergillus flavus</em> Resistance to Stress

Aspergillus section Flavi produce the aflatoxins, secondary metabolites toxic to humans and animals. Why do these fungi produce aflatoxins? They do not have a clear role in pathogenicity or in niche competition. Aspergillus employs a considerable amount of energy to synthesize them: more than 20 enzymatic catalyzes are needed. Within the A. flavus species, all opportunistic pathogens of maize, more than half of the natural population are atoxigenic, indicating that aflatoxins are not so obviously linked to an enhancement of population fitness. The perspective changes in A. parasiticus, pathogen to peanuts, where more than 90% of the natural population produce the four aflatoxins. In this chapter, we aim to discuss our recent hypothesis that aflatoxins act as antioxidants providing more time to Aspergillus to “escape” an exploited substrate, that in the meanwhile is “fully charged” with reactive oxygen species and oxylipins

Fungal lipids.Biosynthesis and signalling during plant-pathogen interaction

Lipids occur in fungi as major constituents of the membrane systems and minor component in the cell wall; they can store energy in the lipid bodies and, in some cases, they can act as intra-extracellular signals. Fungi contain a various set of lipids, including fatty acids, oxylipins, sphingolipids, phospholipids, glycolipids, and sterols. Current studies in lipids suggest their additional role in cell signalling; for instance, host-pathogen exchange lipid signals at the interface during their interaction. This review aims examining those fungal lipid classes involved in the pathogenic interaction with the host plants. The lipid signals may trigger host immune response as well as functioning as virulence factors altering the lipid homeostasis of the host cells

Phytopathogenic fungi threaten the Quercus genus in a coastal forest: detection of fungal diversity by Nanopore Sequencing

Molecular characterization of potential fungal pathogens affecting Quercus spp. is the challenge to limit the expansion of the oak decline. The investigation was made on acorns and on Quercus seedlings grown in a forest nursery. The analysis aims to determine pathogens that can potentially infect seedlings and eventually cause plant death. Fungal detection by molecular tools was performed with two approaches: 1) by culture-dependent fungal profiling followed by Sanger sequencing and 2) by culture-independent fungal profiling using Oxford Nanopore Technology (ONT). In both cases, we sequenced the ITS region of ribosomal DNA. Both methods have proven effective, however, culture-independent methodology proved more advantageous; in fact, using culture independent approach, we were able to identify a larger variety of microorganism putatively involved in the oak decline syndrome. Using culture-dependent approach, several saprophytic fungal species were isolated in vitro and subsequently, characterized by ITS sequencing (Sanger). Nanopore ITS sequencing identified a broader variety of fungal species, with few associated to the oak decline such as Diplodia corticola and Discula quercina. Other fungal species identified like Epicoccum nigrum, Alternaria alternata, Alternaria sp., Lophiostoma corticola and Pleosporales sp. could play an important ecological role as they can change their lifestyles from endophytic to pathogenic, or saprobic, depending on the environmental conditions. Our research provides a phytosanitary protocol to rapidly diagnostic the presence of oak pathogens. Diagnosticated microbial genomes may be used to correlate the potential fungal infections to abiotic stresses

Fatty acid metabolism in Fusarium verticillioides

Fatty acids (FA) are a metabolic energy source and are building blocks of membrane lipids; moreover, they play a role in the signaling among hosts and pathogens. The ascomycete Fusarium verticillioides is a pathogenic fungus of maize; it is a valuable organism for elucidating processes in eukaryotic cell such as fatty acid metabolism. Oxidized FA, i.e. the oxylipins, are important driver of the host-pathogen interaction. Previous studies assessed that their synthesis appear tightly coordinated within the subset of oxylipin-related genes and with the genes controlling the synthesis of FA. We suggest here that the calcium-regulated transcription factor Crz1 (orthologue in F. verticillioides Fvcrz1) is probably involved in the upstream regulation of several genes related to fatty acid and oxylipin synthesis. Through a transcriptomic and metabolomics approach, we analyzed gene expression and FA synthesis in F. verticillioides wild type and mutant strain crz1 under in vitro conditions. Notably, we evaluated the expression of genes related to the FA biosynthesis, transformation, and oxylipins synthesis. Transcriptomic approach was associated to a mass spectrometry analysis to quantify several lipids. Apparently and intriguingly, Crz1 did not affect - significantly - the expression of FA- and oxylipin-related genes but controlled the total amount of FA and oxylipin in F. verticillioides. In fact, FA and oxylipin amount is reduced significantly in crz1 compared to WT strain whilst gene expression resulted almost unscathed. Advances in understanding such processes in F. verticillioides will provide helpful insights to the study of lipid-mediated cross talk within fungus-plant interaction