Seasonal and Spatial Dynamics of Fungal Diversity and Communities in the Intertidal Zones of Qingdao, China.

Intertidal zones comprise diverse habitats and directly suffer from the influences of human activities. Nevertheless, the seasonal fluctuations in fungal diversity and community structure in these areas are not well comprehended. To address this gap, samples of seawater and sediment were collected seasonally from the estuary and swimming beaches of Qingdao's intertidal areas in China and were analyzed using a metabarcoding approach targeting ITS2 rDNA regions. Compared to the seawater community dominated by Ciliophora and Agaricomycetes, the sediment community was rather dominated by Dothideomycetes and Eurotiomycetes. Furthermore, the seawater community shifted with the seasons but not with the locations, while the sediment community shifted seasonally and spatially, with a specific trend showing that Cladosporium, Alternaria, and Aureobasidium occurred predominantly in the estuarine habitats during winter and in the beach habitats during spring. These spatiotemporal shifts in fungal communities' composition were supported by the PERMANOVA test and could be explained partially by the environmental variables checked, including temperature, salinity, and total organic carbon. Unexpectedly, the lowest fungal richness was observed in the summer sediments from two swimming beaches which were attracting a high influx of tourists during summer, leading to a significant anthropogenic influence. Predicted trophic modes of fungal taxa exhibited a seasonal pattern with an abundance of saprotrophic fungi in the summer sediments, positively correlating to the temperature, while the taxa affiliated with symbiotroph and pathotroph-saprotroph occurred abundantly in the winter and spring sediments, respectively. Our results demonstrate the space-time shifts in terms of the fungal community, as well as the trophic modes in the intertidal region, providing in-depth insights into the potential influence of environmental factors and human activity on intertidal mycobiomes.

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (Km = 26 µM) than chloride (Km = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.

Mission Tara Microplastics: a holistic set of protocols and data resources for the field investigation of plastic pollution along the land-sea continuum in Europe.

The Tara Microplastics mission was conducted for 7 months to investigate plastic pollution along nine major rivers in Europe-Thames, Elbe, Rhine, Seine, Loire, Garonne, Ebro, Rhone, and Tiber. An extensive suite of sampling protocols was applied at four to five sites on each river along a salinity gradient from the sea and the outer estuary to downstream and upstream of the first heavily populated city. Biophysicochemical parameters including salinity, temperature, irradiance, particulate matter, large and small microplastics (MPs) concentration and composition, prokaryote and microeukaryote richness, and diversity on MPs and in the surrounding waters were routinely measured onboard the French research vessel Tara or from a semi-rigid boat in shallow waters. In addition, macroplastic and microplastic concentrations and composition were determined on river banks and beaches. Finally, cages containing either pristine pieces of plastics in the form of films or granules, and others containing mussels were immersed at each sampling site, 1 month prior to sampling in order to study the metabolic activity of the plastisphere by meta-OMICS and to run toxicity tests and pollutants analyses. Here, we fully described the holistic set of protocols designed for the Mission Tara Microplastics and promoted standard procedures to achieve its ambitious goals: (1) compare traits of plastic pollution among European rivers, (2) provide a baseline of the state of plastic pollution in the Anthropocene, (3) predict their evolution in the frame of the current European initiatives, (4) shed light on the toxicological effects of plastic on aquatic life, (5) model the transport of microplastics from land towards the sea, and (6) investigate the potential impact of pathogen or invasive species rafting on drifting plastics from the land to the sea through riverine systems.

Metatranscriptomics and metabarcoding reveal spatiotemporal shifts in fungal communities and their activities in Chinese coastal waters.

Fungal communities are diverse and abundant in coastal waters, yet, their ecological roles and adaptations remain largely unknown. To address these gaps, ITS2 metabarcoding and metatranscriptomic analyses were used to capture the whole suite of fungal diversity and their metabolic potential in water column and sediments in the Yellow Sea during August and October 2019. ITS2 metabarcoding described successfully the abundance of Dikarya during August and October at the different examined habitats, but strongly underrepresented or failed to identify other fungal taxa, including zoosporic and early-diverging lineages, that were abundant in the mycobiome as uncovered by metatranscriptomes. Metatranscriptomics also revealed enriched expression of genes annotated to zoosporic fungi (e.g., chytrids) mainly in the surface water column in October. This enriched expression was correlated with the two-fold increase in chlorophyll-a intensity attributed to phytoplanktonic species which are known to be parasitized by chytrids. The concurrent high expression of genes related to calcium signalling and GTPase activity suggested that these metabolic traits facilitate the parasitic lifestyle of chytrids. Similarly, elevated expression of phagosome genes annotated to Rozellomycota, an early-diverging fungal phylum not fully detected with ITS2 metabarcoding, suggested that this taxon utilizes a suite of feeding modes, including phagotrophy in this coastal setting. Our data highlight the necessity of using combined approaches to accurately describe the community structure of coastal mycobiome. We also provide in-depth insights into the fungal ecological roles in coastal waters, and report potential metabolic mechanisms utilized by fungi to cope with environmental stresses that occur during distinct seasonal months in coastal ecosystems.

Bacterial Abundance, Diversity and Activity During Long-Term Colonization of Non-biodegradable and Biodegradable Plastics in Seawater.

The microorganisms living on plastics called "plastisphere" have been classically described as very abundant, highly diverse, and very specific when compared to the surrounding environments, but their potential ability to biodegrade various plastic types in natural conditions have been poorly investigated. Here, we follow the successive phases of biofilm development and maturation after long-term immersion in seawater (7 months) on conventional [fossil-based polyethylene (PE) and polystyrene (PS)] and biodegradable plastics [biobased polylactic acid (PLA) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV), or fossil-based polycaprolactone (PCL)], as well as on artificially aged or non-aged PE without or with prooxidant additives [oxobiodegradable (OXO)]. First, we confirmed that the classical primo-colonization and growth phases of the biofilms that occurred during the first 10 days of immersion in seawater were more or less independent of the plastic type. After only 1 month, we found congruent signs of biodegradation for some bio-based and also fossil-based materials. A continuous growth of the biofilm during the 7 months of observation (measured by epifluorescence microscopy and flow cytometry) was found on PHBV, PCL, and artificially aged OXO, together with a continuous increase in intracellular (3H-leucine incorporation) and extracellular activities (lipase, aminopeptidase, and ß-glucosidase) as well as subsequent changes in biofilm diversity that became specific to each polymer type (16S rRNA metabarcoding). No sign of biodegradation was visible for PE, PS, and PLA under our experimental conditions. We also provide a list of operational taxonomic units (OTUs) potentially involved in the biodegradation of these polymers under natural seawater conditions, such as Pseudohongiella sp. and Marinobacter sp. on PCL, Marinicella litoralis and Celeribacter sp. on PHBV, or Myxococcales on artificially aged OXO. This study opens new routes for a deeper understanding of the polymers' biodegradability in seawaters, especially when considering an alternative to conventional fossil-based plastics.

Environmental factors shaping bacterial, archaeal and fungal community structure in hydrothermal sediments of Guaymas Basin, Gulf of California.

The flanking regions of Guaymas Basin, a young marginal rift basin located in the Gulf of California, are covered with thick sediment layers that are hydrothermally altered due to magmatic intrusions. To explore environmental controls on microbial community structure in this complex environment, we analyzed site- and depth-related patterns of microbial community composition (bacteria, archaea, and fungi) in hydrothermally influenced sediments with different thermal conditions, geochemical regimes, and extent of microbial mats. We compared communities in hot hydrothermal sediments (75-100°C at ~40 cm depth) covered by orange-pigmented Beggiatoaceae mats in the Cathedral Hill area, temperate sediments (25-30°C at ~40 cm depth) covered by yellow sulfur precipitates and filamentous sulfur oxidizers at the Aceto Balsamico location, hot sediments (>115°C at ~40 cm depth) with orange-pigmented mats surrounded by yellow and white mats at the Marker 14 location, and background, non-hydrothermal sediments (3.8°C at ~45 cm depth) overlain with ambient seawater. Whereas bacterial and archaeal communities are clearly structured by site-specific in-situ thermal gradients and geochemical conditions, fungal communities are generally structured by sediment depth. Unexpectedly, chytrid sequence biosignatures are ubiquitous in surficial sediments whereas deeper sediments contain diverse yeasts and filamentous fungi. In correlation analyses across different sites and sediment depths, fungal phylotypes correlate to each other to a much greater degree than Bacteria and Archaea do to each other or to fungi, further substantiating that site-specific in-situ thermal gradients and geochemical conditions that control bacteria and archaea do not extend to fungi.

Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity.

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

Deep-sea hydrothermal vent sediments reveal diverse fungi with antibacterial activities.

Relatively little is known about the diversity of fungi in deep-sea, hydrothermal sediments. Less thoroughly explored environments are likely untapped reservoirs of unique biodiversity with the potential to augment our current arsenal of microbial compounds with biomedical and/or industrial applications. In this study, we applied traditional culture-based methods to examine a subset of the morphological and phylogenetic diversity of filamentous fungi and yeasts present in 11 hydrothermally influenced sediment samples collected from eight sites on the seafloor of Guaymas Basin, Mexico. A total of 12 unique isolates affiliating with Ascomycota and Basidiomycota were obtained and taxonomically identified on the basis of morphological features and analyses of marker genes including actin, ß-tubulin, small subunit ribosomal DNA (18S rRNA), internal transcribed spacer (ITS) and large subunit ribosomal DNA (26S rRNA) D1/D2 domain sequences (depending on taxon). A total of 11 isolates possess congeners previously detected in, or recovered from, deep-sea environments. A total of seven isolates exhibited antibacterial activity against human bacterial pathogens Staphylococcus aureus ATCC-35556 and/or Escherichia coli ATCC-25922. This first investigation suggests that hydrothermal environments may serve as promising reservoirs of much greater fungal diversity, some of which may produce biomedically useful metabolites.

Seasonal dynamics of mycoplankton in the Yellow Sea reflect the combined effect of riverine inputs and hydrographic conditions.

Little is known about how multiple factors including land-based inputs and ocean currents affect the spatiotemporal distribution of the mycoplankton in coastal regions. To explore the seasonal changes of mycoplanktonic communities and potential environmental drivers, we collected water samples from the Yellow Sea, used here as a model for subtropical sea habitats, in different seasons over two years. Compared with winter and spring, summer exhibited higher levels of fungal richness and community heterogeneity in the water column. The seasonal shifts in mycoplankton diversity and community composition were mainly ascribed to freshwater inputs, the Cold Water Mass and invasion of the Yellow Sea Warm Current. Among the physicochemical variables tested, temperature was the primary determinant of fungal diversity and showed contrasting influences on fungal richness in the surface and bottom waters during summer. In addition, we provide evidence for the community similarity and dissolved nutrients of different water bodies to highlight the potential origin of the Cold Water Mass. Our findings bring new understanding on the factors determining the dynamics of mycoplankton communities by modelling the influence of physicochemical variables and tracking the geographical distribution of certain fungal taxa.

Meta-omics highlights the diversity, activity and adaptations of fungi in deep oceanic crust.

The lithified oceanic crust, lower crust gabbros in particular, has remained largely unexplored by microbiologists. Recently, evidence for heterogeneously distributed viable and transcriptionally active autotrophic and heterotrophic microbial populations within low-biomass communities was found down to 750 m below the seafloor at the Atlantis Bank Gabbro Massif, Indian Ocean. Here, we report on the diversity, activity and adaptations of fungal communities in the deep oceanic crust from ~10 to 780 mbsf by combining metabarcoding analyses with mid/high-throughput culturing approaches. Metabarcoding along with culturing indicate a low diversity of viable fungi, mostly affiliated to ubiquitous (terrestrial and aquatic environments) taxa. Ecophysiological analyses coupled with metatranscriptomics point to viable and transcriptionally active fungal populations engaged in cell division, translation, protein modifications and other vital cellular processes. Transcript data suggest possible adaptations for surviving in the nutrient-poor, lithified deep biosphere that include the recycling of organic matter. These active communities appear strongly influenced by the presence of cracks and veins in the rocks where fluids and resulting rock alteration create micro-niches.

Recycling and metabolic flexibility dictate life in the lower oceanic crust.

The lithified lower oceanic crust is one of Earth's last biological frontiers as it is difficult to access. It is challenging for microbiota that live in marine subsurface sediments or igneous basement to obtain sufficient carbon resources and energy to support growth1-3 or to meet basal power requirements4 during periods of resource scarcity. Here we show how limited and unpredictable sources of carbon and energy dictate survival strategies used by low-biomass microbial communities that live 10-750 m below the seafloor at Atlantis Bank, Indian Ocean, where Earth's lower crust is exposed at the seafloor. Assays of enzyme activities, lipid biomarkers, marker genes and microscopy indicate heterogeneously distributed and viable biomass with ultralow cell densities (fewer than 2,000 cells per cm3). Expression of genes involved in unexpected heterotrophic processes includes those with a role in the degradation of polyaromatic hydrocarbons, use of polyhydroxyalkanoates as carbon-storage molecules and recycling of amino acids to produce compounds that can participate in redox reactions and energy production. Our study provides insights into how microorganisms in the plutonic crust are able to survive within fractures or porous substrates by coupling sources of energy to organic and inorganic carbon resources that are probably delivered through the circulation of subseafloor fluids or seawater.

Insights into fungal diversity of a shallow-water hydrothermal vent field at Kueishan Island, Taiwan by culture-based and metabarcoding analyses.

This paper reports the diversity of fungi associated with substrates collected at a shallow hydrothermal vent field at Kueishan Island, Taiwan, using both culture-based and metabarcoding methods. Culture of fungi from yellow sediment (with visible sulfur granules), black sediment (no visible sulfur granules), the vent crab Xenograpsus testudinatus, seawater and, animal egg samples resulted in a total of 94 isolates. Species identification based on the internal transcribed spacer regions of the rDNA revealed that the yellow sediment samples had the highest species richness with 25 species, followed by the black sediment (23) and the crab (13). The Ascomycota was dominant over the Basidiomycota; the dominant orders were Agaricales, Capnodiales, Eurotiales, Hypocreales, Pleosporales, Polyporales and Xylariales. Hortaea werneckii was the only common fungus isolated from the crab, seawater, yellow and black sediment samples. The metabarcoding analysis amplifying a small fragment of the rDNA (from 18S to 5.8S) recovered 7-27 species from the black sediment and 12-27 species from the yellow sediment samples and all species belonged to the Ascomycota and the Basidiomycota. In the yellow sediments, the dominant order was Pleosporales and this order was also dominant in the black sediment together with Sporidiobolales. Based on the results from both methods, 54 and 49 species were found in the black and yellow sediments, respectively. Overall, a higher proportion of Ascomycota (~70%) over Basidiomycota was recovered in the yellow sediment and the two phyla were equally abundant in the black sediment. The top five dominant fungal orders in descending order based on species richness were Pleosporales>Eurotiales>Polyporales>Hypocreales>Capnodiales in the black sediment samples, and Polyporales>Pleosporales>Eurotiales>Capnodiales>Hypocreales in the yellow sediment samples. This study is the first to observe a high diversity of fungi associated with various substrates at a marine shallow water hydrothermal vent ecosystem. While some fungi found in this study were terrestrial species and their airborne spores might have been deposited into the marine sediment, several pathogenic fungi of animals, including Acremonium spp., Aspergillus spp., Fusarium spp., Malassezia spp., Hortaea werneckii, Parengyodontium album, and Westerdykella dispersa, were recovered suggesting that these fungi may be able to cause diseases of marine animals.

Fungal Community Composition and Potential Depth-Related Driving Factors Impacting Distribution Pattern and Trophic Modes from Epi- to Abyssopelagic Zones of the Western Pacific Ocean.

Fungi play an important role in cycling organic matter and nutrients in marine ecosystems. However, the distribution of fungal communities in the ocean, especially the vertical distribution along depth in the water column, remained poorly understood. Here, we assess the depth-related distribution pattern of fungal communities along the water column from epi- to abyssopelagic zones of the Western Pacific Ocean using internal transcribed spacer 2 (ITS2) metabarcoding. Majority of the assigned OTUs were affiliated to Ascomycota, followed by three other minor phyla (Basidiomycota, Chytridiomycota, and Mucoromycota). The epipelagic zone harbored a higher OTU richness with distinct fungal communities as compared with meso-, bathy-, and abyssopelagic zones. Across the whole water column, depth appears as a key parameter for both fungal α- and ß-diversity. However, when the dataset was split into the upper (5-500 m) and deeper (below 500 m) layers, no significant correlation was observed between depth and community compositions. In the upper layer, temperature and dissolved oxygen were recognized as the primary environmental factors shaping fungal α- and ß- diversity. By parsing fungal OTUs into ecological categories, multi-trophic mode of nutrition was found to be more prevalent with increasing depth, suggesting a potential adaptation to the extreme conditions of the deep sea. This study provides new and meaningful information on the depth-stratified fungal diversity, community structure, and putative ecological roles in the open sea.

Fungi in the Marine Environment: Open Questions and Unsolved Problems.

Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.

Highlighting patterns of fungal diversity and composition shaped by ocean currents using the East China Sea as a model.

How ocean currents shape fungal transport, dispersal and more broadly fungal biogeography remains poorly understood. The East China Sea (ECS) is a complex and dynamic habitat with different water masses blending microbial communities. The internal transcribed spacer 2 region of fungal rDNA was analysed in water and sediment samples directly collected from the coastal (CWM), Kuroshio (KSWM), Taiwan warm (TWM) and the shelf mixed water mass (MWM), coupled with hydrographic properties measurements, to determine how ocean currents impact the fungal community composition. Almost 9k fungal operational taxonomic units (OTUs) spanning six phyla, 25 known classes, 102 orders and 694 genera were obtained. The typical terrestrial and freshwater fungal genus, Byssochlamys, was dominant in the CWM, while increasing abundance of a specific OTU affiliated with Aspergillus was revealed from coastal to open ocean water masses (TWM and KSWM). Compared with water samples, sediment harboured an increased diversity with distinct fungal communities. The proximity of the Yangtze and Qiantang estuaries homogenizes the surface water and sediment communities. A significant influence of ocean currents on community structure was found, which is believed to reduce proportionally the variation explained by environmental parameters at the scale of the total water masses. Dissolved oxygen and depth were identified as the major parameters structuring the fungal community. Our results indicate that passive fungal dispersal driven by ocean currents and river run-off, in conjunction with the distinct hydrographic conditions of individual water masses, shapes the fungal community composition and distribution pattern in the ECS.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

Bioactive Metabolites from the Deep Subseafloor Fungus Oidiodendron griseum UBOCC-A-114129.

Four bioactive compounds have been isolated from the fungus Oidiodendron griseum UBOCC-A-114129 cultivated from deep subsurface sediment. They were structurally characterized using a combination of LC-MS/MS and NMR analyses as fuscin and its derivatives (dihydrofuscin, dihydrosecofuscin, and secofuscin) and identified as polyketides. Albeit those compounds were already obtained from terrestrial fungi, this is the first report of their production by an Oidiodendron species and by the deepest subseafloor isolate ever studied for biological activities. We report a weak antibacterial activity of dihydrosecofuscin and secofuscin mainly directed against Gram-positive bacteria (Minimum Inhibitory Concentration (MIC) equal to Minimum Bactericidal Concentration (MBC), in the range of 100 µg/mL). The activity on various protein kinases was also analyzed and revealed a significant inhibition of CDC2-like kinase-1 (CLK1) by dihysecofuscin.

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.

Fungal and Prokaryotic Activities in the Marine Subsurface Biosphere at Peru Margin and Canterbury Basin Inferred from RNA-Based Analyses and Microscopy.

The deep sedimentary biosphere, extending 100s of meters below the seafloor harbors unexpected diversity of Bacteria, Archaea, and microbial eukaryotes. Far less is known about microbial eukaryotes in subsurface habitats, albeit several studies have indicated that fungi dominate microbial eukaryotic communities and fungal molecular signatures (of both yeasts and filamentous forms) have been detected in samples as deep as 1740 mbsf. Here, we compare and contrast fungal ribosomal RNA gene signatures and whole community metatranscriptomes present in sediment core samples from 6 and 95 mbsf from Peru Margin site 1229A and from samples from 12 and 345 mbsf from Canterbury Basin site U1352. The metatranscriptome analyses reveal higher relative expression of amino acid and peptide transporters in the less nutrient rich Canterbury Basin sediments compared to the nutrient rich Peru Margin, and higher expression of motility genes in the Peru Margin samples. Higher expression of genes associated with metals transporters and antibiotic resistance and production was detected in Canterbury Basin sediments. A poly-A focused metatranscriptome produced for the Canterbury Basin sample from 345 mbsf provides further evidence for active fungal communities in the subsurface in the form of fungal-associated transcripts for metabolic and cellular processes, cell and membrane functions, and catalytic activities. Fungal communities at comparable depths at the two geographically separated locations appear dominated by distinct taxa. Differences in taxonomic composition and expression of genes associated with particular metabolic activities may be a function of sediment organic content as well as oceanic province. Microscopic analysis of Canterbury Basin sediment samples from 4 and 403 mbsf produced visualizations of septate fungal filaments, branching fungi, conidiogenesis, and spores. These images provide another important line of evidence supporting the occurrence and activity of fungi in the deep subseafloor biosphere.