Fungal dye-decolorizing peroxidase diversity: roles in either intra- or extracellular processes.

Fungal dye-decolorizing peroxidases (DyPs) have found applications in the treatment of dye-contaminated industrial wastes or to improve biomass digestibility. Their roles in fungal biology are uncertain, although it has been repeatedly suggested that they could participate in lignin degradation and/or modification. Using a comprehensive set of 162 fully sequenced fungal species, we defined seven distinct fungal DyP clades on basis of a sequence similarity network. Sequences from one of these clades clearly diverged from all others, having on average the lower isoelectric points and hydropathy indices, the highest number of N-glycosylation sites, and N-terminal sequence peptides for secretion. Putative proteins from this clade are absent from brown-rot and ectomycorrhizal species that have lost the capability of degrading lignin enzymatically. They are almost exclusively present in white-rot and other saprotrophic Basidiomycota that digest lignin enzymatically, thus lending support for a specific role of DyPs from this clade in biochemical lignin modification. Additional nearly full-length fungal DyP genes were isolated from the environment by sequence capture by hybridization; they all belonged to the clade of the presumably secreted DyPs and to another related clade. We suggest focusing our attention on the presumably intracellular DyPs from the other clades, which have not been characterized thus far and could represent enzyme proteins with novel catalytic properties. KEY POINTS: • A fungal DyP phylogeny delineates seven main sequence clades. • Putative extracellular DyPs form a single clade of Basidiomycota sequences. • Extracellular DyPs are associated to white-rot fungi.

The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization.

Mutualistic plant-associated fungi are recognized as important drivers in plant evolution, diversity, and health. The discovery that mycoviruses can take part and play important roles in symbiotic tripartite interactions has prompted us to study the viromes associated with a collection of ericoid and orchid mycorrhizal (ERM and ORM, respectively) fungi. Our study, based on high-throughput sequencing of transcriptomes (RNAseq) from fungal isolates grown in axenic cultures, revealed in both ERM and ORM fungi the presence of new mycoviruses closely related to already classified virus taxa, but also new viruses that expand the boundaries of characterized RNA virus diversity to previously undescribed evolutionary trajectories. In ERM fungi, we provide first evidence of a bipartite virus, distantly related to narnaviruses, that splits the RNA-dependent RNA polymerase (RdRP) palm domain into two distinct proteins, encoded by each of the two segments. Furthermore, in one isolate of the ORM fungus Tulasnella spp. we detected a 12 kb genomic fragment coding for an RdRP with features of bunyavirus-like RdRPs. However, this 12 kb genomic RNA has the unique features, for Bunyavirales members, of being tri-cistronic and carrying ORFs for the putative RdRP and putative nucleocapsid in ambisense orientation on the same genomic RNA. Finally, a number of ORM fungal isolates harbored a group of ambisense bicistronic viruses with a genomic size of around 5 kb, where we could identify a putative RdRP palm domain that has some features of plus strand RNA viruses; these new viruses may represent a new lineage in the Riboviria, as they could not be reliably assigned to any of the branches in the recently derived monophyletic tree that includes most viruses with an RNA genome.

Metabarcoding on both environmental DNA and RNA highlights differences between fungal communities sampled in different habitats.

In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based (total) and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either "active" or "total" microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone.

Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

The Dark Side of Orchid Symbiosis: Can Tulasnella calospora Decompose Host Tissues?

Photosynthetic orchids associate with mycorrhizal fungi that can be mostly ascribed to the "rhizoctonia" species complex. Rhizoctonias' phylogenetic diversity covers a variety of ecological/nutritional strategies that include, beside the symbiosis establishment with host plants, endophytic and pathogenic associations with non-orchid plants or saprotrophic soil colonization. In addition, orchid mycorrhizal fungi (OMF) that establish a symbiotic relationship with an orchid host can later proliferate in browning and rotting orchid tissues. Environmental triggers and molecular mechanisms governing the switch leading to either a saprotrophic or a mycorrhizal behavior in OMF remain unclear. As the sequenced OMF genomes feature a wide range of genes putatively involved in the degradation of plant cell wall (PCW) components, we tested if these transitions may be correlated with a change in the expression of some PCW degrading enzymes. Regulation of several genes encoding PCW degrading enzymes was evaluated during saprotrophic growth of the OMF Tulasnella calospora on different substrates and under successful and unsuccessful mycorrhizal symbioses. Fungal gene expression in planta was investigated in two orchid species, the terrestrial Mediterranean Serapias vomeracea and the epiphytic tropical Cattleya purpurata. Although we only tested a subset of the CAZyme genes identified in the T. calospora genome, and we cannot exclude therefore a role for different CAZyme families or members inside a family, the results showed that the degradative potential of T. calospora is finely regulated during saprotrophic growth and in symbiosis, often with a different regulation in the two orchid species. These data pose novel questions about the role of fungal PCW degrading enzymes in the development of unsuccessful and successful interactions.

Soil bacterial networks are less stable under drought than fungal networks.

Soil microbial communities play a crucial role in ecosystem functioning, but it is unknown how co-occurrence networks within these communities respond to disturbances such as climate extremes. This represents an important knowledge gap because changes in microbial networks could have implications for their functioning and vulnerability to future disturbances. Here, we show in grassland mesocosms that drought promotes destabilising properties in soil bacterial, but not fungal, co-occurrence networks, and that changes in bacterial communities link more strongly to soil functioning during recovery than do changes in fungal communities. Moreover, we reveal that drought has a prolonged effect on bacterial communities and their co-occurrence networks via changes in vegetation composition and resultant reductions in soil moisture. Our results provide new insight in the mechanisms through which drought alters soil microbial communities with potential long-term consequences, including future plant community composition and the ability of aboveground and belowground communities to withstand future disturbances.

RNA extraction from decaying wood for (meta)transcriptomic analyses.

Wood decomposition is a key step of the terrestrial carbon cycle and is of economic importance. It is essentially a microbiological process performed by fungi and to an unknown extent by bacteria. To gain access to the genes expressed by the diverse microbial communities participating in wood decay, we developed an RNA extraction protocol from this recalcitrant material rich in polysaccharides and phenolic compounds. This protocol was implemented on 22 wood samples representing as many tree species from 11 plant families in the Angiosperms and Gymnosperms. RNA was successfully extracted from all samples and converted into cDNAs from which were amplified both fungal and bacterial protein coding genes, including genes encoding hydrolytic enzymes participating in lignocellulose hydrolysis. This protocol applicable to a wide range of decomposing wood types represents a first step towards a metatranscriptomic analysis of wood degradation under natural conditions.

Cell-specific expression of plant nutrient transporter genes in orchid mycorrhizae.

Orchid mycorrhizal protocorms and roots are heterogeneous structures composed of different plant cell-types, where cells colonized by intracellular fungal coils (the pelotons) are close to non-colonized plant cells. Moreover, the fungal coils undergo rapid turnover inside the colonized cells, so that plant cells containing coils at different developmental stages can be observed in the same tissue section. Here, we have investigated by laser microdissection (LMD) the localization of specific plant gene transcripts in different cell-type populations collected from mycorrhizal protocorms and roots of the Mediterranean orchid Serapias vomeracea colonized by Tulasnella calospora. RNAs extracted from the different cell-type populations have been used to study plant gene expression, focusing on genes potentially involved in N uptake and transport and previously identified as up-regulated in symbiotic protocorms. Results clearly showed that some plant N transporters are differentially expressed in cells containing fungal coils at different developmental stages, as well as in non-colonized cells, and allowed the identification of new functional markers associated to coil-containing cells.

Soil networks become more connected and take up more carbon as nature restoration progresses.

Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered.

Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands.

Mycorrhizal fungi are essential for the survival of orchid seedlings under natural conditions. The distribution of these fungi in soil can constrain the establishment and resulting spatial arrangement of orchids at the local scale, but the actual extent of occurrence and spatial patterns of orchid mycorrhizal (OrM) fungi in soil remain largely unknown. We addressed the fine-scale spatial distribution of OrM fungi in two orchid-rich Mediterranean grasslands by means of high-throughput sequencing of fungal ITS2 amplicons, obtained from soil samples collected either directly beneath or at a distance from adult Anacamptis morio and Ophrys sphegodes plants. Like ectomycorrhizal and arbuscular mycobionts, OrM fungi (tulasnelloid, ceratobasidioid, sebacinoid and pezizoid fungi) exhibited significant horizontal spatial autocorrelation in soil. However, OrM fungal read numbers did not correlate with distance from adult orchid plants, and several of these fungi were extremely sporadic or undetected even in the soil samples containing the orchid roots. Orchid mycorrhizal 'rhizoctonias' are commonly regarded as unspecialized saprotrophs. The sporadic occurrence of mycobionts of grassland orchids in host-rich stands questions the view of these mycorrhizal fungi as capable of sustained growth in soil.

Fungal and plant gene expression in the Tulasnella calospora-Serapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas.

Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer. We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources. Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface. This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments.

Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists.

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.

Temporal variation in mycorrhizal diversity and carbon and nitrogen stable isotope abundance in the wintergreen meadow orchid Anacamptis morio.

Many adult orchids, especially photoautotrophic species, associate with a diverse range of mycorrhizal fungi, but little is known about the temporal changes that might occur in the diversity and functioning of orchid mycorrhiza during vegetative and reproductive plant growth. Temporal variations in the spectrum of mycorrhizal fungi and in stable isotope natural abundance were investigated in adult plants of Anacamptis morio, a wintergreen meadow orchid. Anacamptis morio associated with mycorrhizal fungi belonging to Tulasnella, Ceratobasidium and a clade of Pezizaceae (Ascomycetes). When a complete growing season was investigated, multivariate analyses indicated significant differences in the mycorrhizal fungal community. Among fungi identified from manually isolated pelotons, Tulasnella was more common in autumn and winter, the pezizacean clade was very frequent in spring, and Ceratobasidium was more frequent in summer. By contrast, relatively small variations were found in carbon (C) and nitrogen (N) stable isotope natural abundance, A. morio samples showing similar (15)N enrichment and (13)C depletion at the different sampling times. These observations suggest that, irrespective of differences in the seasonal environmental conditions, the plant phenological stages and the associated fungi, the isotopic content in mycorrhizal A. morio remains fairly constant over time.

Contrasting land uses in Mediterranean agro-silvo-pastoral systems generated patchy diversity patterns of vascular plants and below-ground microorganisms.

The aims of this paper were (i) to define how contrasting land uses affected plant biodiversity in Mediterranean agro-silvo-pastoral-systems across a gradient of disturbance regimes: cork oak forests, secondary grasslands, hay crops, grass covered vineyards, tilled vineyards; (ii) to determine whether these patterns mirrored those of below-ground microorganisms and whether the components of γ-diversity followed a similar model. The disturbance regimes affected plant assemblage composition. Species richness decreased with increasing land use intensity, the Shannon index showed the highest values in grasslands and hay crops. Plant assemblage composition patterns mirrored those of Basidiomycota and Ascomycota. Richness in Basidiomycota, denitrifying bacteria and microbial biomass showed the same trend as that observed for vascular plant richness. The Shannon index pattern of below-ground microorganisms was different from that of plants. The plant γ-diversity component model weakly mirrored those of Ascomycota. Patchy diversity patterns suggest that the maintenance of contrasting land uses associated with different productions typical of agro-silvo-pastoral-systems can guarantee the conservation of biodiversity.

Plant and fungal gene expression in mycorrhizal protocorms of the orchid Serapias vomeracea colonized by Tulasnella calospora.

Little is known on the molecular bases of plant-fungal interactions in orchid mycorrhiza. We developed a model system to investigate gene expression in mycorrhizal protocorms of Serapias vomeracea colonised by Tulasnella calospora. Our recent results with a small panel of genes as indicators of plant response to mycorrhizal colonization indicate that genes related with plant defense were not significantly up-regulated in mycorrhizal tissues. Here, we used laser microdissection to investigate whether expression of some orchid genes was restricted to specific cell types. Results showed that SvNod1, a S. vomeracea nodulin-like protein containing a plastocyanin-like domain, is expressed only in protocorm cells containing intracellular fungal hyphae. In addition, we investigated a family of fungal zinc metallopeptidases (M36). This gene family has expanded in the T. calospora genome and RNA-Seq experiments indicate that some members of the M36 metallopeptidases family are differentially regulated in orchid mycorrhizal protocorms.

Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant-fungus relationship.

Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.

Cryopreservation of orchid mycorrhizal fungi: a tool for the conservation of endangered species.

The effectiveness of cryopreservation at --80 °C on orchid mycorrhizal fungi was assessed by testing the symbiotic ability of ten fungal isolates following cryo-storage for 10-24 months. The results obtained prove the efficacy of the method, thus providing a valuable tool for ex situ conservation.

Fungal diversity is not determined by mineral and chemical differences in serpentine substrates.

The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter.

Unravelling soil fungal communities from different Mediterranean land-use backgrounds.

BACKGROUND: Fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological services as decomposers, plant mutualists and pathogens. The Mediterranean area is a biodiversity hotspot that is increasingly threatened by intense land use. Therefore, to achieve a balance between conservation and human development, a better understanding of the impact of land use on the underlying fungal communities is needed. METHODOLOGY/PRINCIPAL FINDINGS: We used parallel pyrosequencing of the nuclear ribosomal its regions to characterize the fungal communities in five soils subjected to different anthropogenic impact in a typical mediterranean landscape: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards. Marked differences in the distribution of taxon assemblages among the different sites and communities were found. Data analyses consistently indicated a sharp distinction of the fungal community of the cork oak forest soil from those described in the other soils. Each soil showed features of the fungal assemblages retrieved which can be easily related to the above-ground settings: ectomycorrhizal phylotypes were numerous in natural sites covered by trees, but were nearly completely missing from the anthropogenic and grass-covered sites; similarly, coprophilous fungi were common in grazed sites. CONCLUSIONS/SIGNIFICANCE: Data suggest that investigation on the below-ground fungal community may provide useful elements on the above-ground features such as vegetation coverage and agronomic procedures, allowing to assess the cost of anthropogenic land use to hidden diversity in soil. Datasets provided in this study may contribute to future searches for fungal bio-indicators as biodiversity markers of a specific site or a land-use degree.