Fungal Patterns from Soils in Madagascar: an Insight from Maromizaha Forest (Evergreen Humid Forest) to Outside (Deciduous Forest).

Soil fungal diversity was studied by next-generation sequencing and compared in two different Malagasy ecosystems, the first a New Protected Area (Maromizaha NAP) that is a rich humid evergreen forest and the second a degraded and declined deciduous forest (Andaravina) whose area has been also eroded. Both areas, however, have comparable annual rainfalls and soil pH values. So it was of interest to examine the soil fungal diversity in each system and compare them. We detected 1,817,658 reads representing Ascomycota, which were dominant in both habitats (55.9%), followed by unidentified fungi (21.5%), Basidiomycota (12.7%) and Mortierellomycota (6.7%), with Mucoromycota, Chytridiomycota, Glomeromycota and other phyla accounting for less than 5% in total. In detail, 1,142 OTUs out of 1,368 constitute the common core shared by both sampling areas, which are characterized by tropical climate, whereas 185 are Maromizaha specific and 41 Andaravina specific. The most represented guilds involve fungi related to saprotrophic behaviour, with a greater tendency towards pathotrophic mode. A significant variability in terms of richness and abundance is present within Maromizaha, which is a heterogeneous environment for fungi but also for plant composition, as it emerged from the vegetational survey of the investigated sites. A few fungal sequences match taxa from Madagascar, highlighting the scarce representativeness of fungi from this island in the fungal databases and their still low knowledge. Enlarging studies in Madagascar will help not only to unravel its largely unknown fungal biodiversity but also to give a contribution for studies on the reconstruction of the diversity of soil fungi worldwide.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Early signalling processes in roots play a crucial role in the differential salt tolerance in contrasting Chenopodium quinoa accessions.

Significant variation in epidermal bladder cell (EBC) density and salt tolerance (ST) exists amongst quinoa accessions, suggesting that salt sequestration in EBCs is not the only mechanism conferring ST in this halophyte. In order to reveal other traits that may operate in tandem with salt sequestration in EBCs and whether these additional tolerance mechanisms acted mainly at the root or shoot level, two quinoa (Chenopodium quinoa) accessions with contrasting ST and EBC densities (Q30, low ST with high EBC density versus Q68, with high ST and low EBC density) were studied. The results indicate that responses in roots, rather than in shoots, contributed to the greater ST in the accession with low EBC density. In particular, the tolerant accession had improved root plasma membrane integrity and K+ retention in the mature root zone in response to salt. Furthermore, superior ST in the tolerant Q68 was associated with faster and root-specific H2O2 accumulation and reactive oxygen species-induced K+ and Ca2+ fluxes in the root apex within 30 min after NaCl application. This was found to be associated with the constitutive up-regulation of the membrane-localized receptor kinases regulatory protein FERONIA in the tolerant accession. Taken together, this study shows that differential root signalling events upon salt exposure are essential for the halophytic quinoa; the failure to do this limits quinoa adaptation to salinity, independently of salt sequestration in EBCs.

Different patterns in root and soil fungal diversity drive plant productivity of the desert truffle Terfezia claveryi in plantation.

The desert truffle Terfezia claveryi is one of the few mycorrhizal fungi currently in cultivation in semiarid and arid areas. Agroclimatic parameters seem to affect its annual yield, but there is no information on the influence of biotic factors. In this study, fungal diversity was analysed by high-throughput sequencing of the ITS2 rDNA region from soil and root samples to compare productive and non-productive mycorrhizal plants in a 4-years old plantation (Murcia, Spain). The fungal metaprofile was dominated by Ascomycota phylum. Desert truffle productivity was driven by different patterns of fungal species composition in soil (species replacement) and root (species richness differences). Moreover, positive associations for ectomycorrhizal and negative for arbuscular mycorrhizal guilds were found in productive roots, and positive associations for fungal parasite-plant pathogen guild in non-productive ones. Soil samples were dominated by pathotroph and saprotroph trophic modes, showing positive associations for Aureobasidium pullulans and Alternaria sp. in productive areas, and positive associations for Fusarium sp. and Mortierella sp. were found in non-productive soils. Finally, some significant OTUs were identified and associated to ascocarp producing patches, which could serve as predictive and location markers of desert truffle production.

Early responses to salt stress in quinoa genotypes with opposite behavior.

Soil salinity is among the major abiotic stresses that plants must cope with, mainly in arid and semiarid regions. The tolerance to high salinity is an important agronomic trait to sustain food production. Quinoa is a halophytic annual pseudo-cereal species with high nutritional value that can secrete salt out of young leaves in external non-glandular cells called epidermal bladder cells (EBC). Previous work showed high salt tolerance, but low EBC density was associated with an improved response in the early phases of salinity stress, mediated by tissue-tolerance traits mainly in roots. We compared the transcript profiling of two quinoa genotypes with contrasting salt tolerance patterning to identify the candidate genes involved in the differentially early response among genotypes. The transcriptome profiling, supported by in vitro physiological analyses, provided insights into the early-stage molecular mechanisms, both at the shoot and root level, based on the sensitive/tolerance traits. Results showed the presence of numerous differentially expressed genes among genotypes, tissues, and treatments, with genes involved in hormonal and stress response upregulated mainly in the sensitive genotype, suggesting that tolerance may be correlated to restricted changes in gene expression, at least after a short salt stress. These data, showing constitutive differences between the two genotypes, represent a solid basis for further studies to characterize the salt tolerance traits. Additionally, new information provided by this work might be useful for the development of plant breeding or genome engineering programs in quinoa.

At the nexus of three kingdoms: the genome of the mycorrhizal fungus Gigaspora margarita provides insights into plant, endobacterial and fungal interactions.

As members of the plant microbiota, arbuscular mycorrhizal fungi (AMF, Glomeromycotina) symbiotically colonize plant roots. AMF also possess their own microbiota, hosting some uncultivable endobacteria. Ongoing research has revealed the genetics underlying plant responses to colonization by AMF, but the fungal side of the relationship remains in the dark. Here, we sequenced the genome of Gigaspora margarita, a member of the Gigasporaceae in an early diverging group of the Glomeromycotina. In contrast to other AMF, G. margarita may host distinct endobacterial populations and possesses the largest fungal genome so far annotated (773.104 Mbp), with more than 64% transposable elements. Other unique traits of the G. margarita genome include the expansion of genes for inorganic phosphate metabolism, the presence of genes for production of secondary metabolites and a considerable number of potential horizontal gene transfer events. The sequencing of G. margarita genome reveals the importance of its immune system, shedding light on the evolutionary pathways that allowed early diverging fungi to interact with both plants and bacteria.

Long-Term Impact of Chemical and Alternative Fungicides Applied to Grapevine cv Nebbiolo on Berry Transcriptome.

Viticulture is one of the horticultural systems in which antifungal treatments can be extremely frequent, with substantial economic and environmental costs. New products, such as biofungicides, resistance inducers and biostimulants, may represent alternative crop protection strategies respectful of the environmental sustainability and food safety. Here, the main purpose was to evaluate the systemic molecular modifications induced by biocontrol products as laminarin, resistance inducers (i.e., fosetyl-Al and potassium phosphonate), electrolyzed water and a standard chemical fungicide (i.e., metiram), on the transcriptomic profile of 'Nebbiolo' grape berries at harvest. In addition to a validation of the sequencing data through real-time polymerase chain reaction (PCR), for the first-time the expression of some candidate genes in different cell-types of berry skin (i.e., epidermal and hypodermal layers) was evaluated using the laser microdissection approach. Results showed that several considered antifungal treatments do not strongly affect the berry transcriptome profile at the end of season. Although some treatments do not activate long lasting molecular defense priming features in berry, some compounds appear to be more active in long-term responses. In addition, genes differentially expressed in the two-cell type populations forming the berry skin were found, suggesting a different function for the two-cell type populations.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

The virome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals the first report of DNA fragments corresponding to replicating non-retroviral RNA viruses in fungi.

Arbuscular Mycorrhizal Fungi (AMF) are key components of the plant microbiota. AMF genetic complexity is increased by the presence of endobacteria, which live inside many species. A further component of such complexity is the virome associated to AMF, whose knowledge is still very limited. Here, by exploiting transcriptomic data we describe the virome of Gigaspora margarita. A BLAST search for viral RNA-dependent RNA polymerases sequences allowed the identification of four mitoviruses, one Ourmia-like narnavirus, one Giardia-like virus, and two sequences related to Fusarium graminearum mycoviruses. Northern blot and RT-PCR confirmed the authenticity of all the sequences with the exception of the F. graminearum-related ones. All the mitoviruses are replicative and functional since both positive strand and negative strand RNA are present. The abundance of the viral RNA molecules is not regulated by the presence or absence of Candidatus Glomeribacter gigasporarum, the endobacterium hosted by G. margarita, with the exception of the Ourmia-like sequence which is absent in bacteria-cured spores. In addition, we report, for the first time, DNA fragments corresponding to mitovirus sequences associated to the presence of viral RNA. These sequences are not integrated in the mitochondrial DNA and preliminary evidence seems to exclude integration in the nuclear genome.

Native soils with their microbiotas elicit a state of alert in tomato plants.

Several studies have investigated soil microbial biodiversity, but understanding of the mechanisms underlying plant responses to soil microbiota remains in its infancy. Here, we focused on tomato (Solanum lycopersicum), testing the hypothesis that plants grown on native soils display different responses to soil microbiotas. Using transcriptomics, proteomics, and biochemistry, we describe the responses of two tomato genotypes (susceptible or resistant to Fusarium oxysporum f. sp. lycopersici) grown on an artificial growth substrate and two native soils (conducive and suppressive to Fusarium). Native soils affected tomato responses by modulating pathways involved in responses to oxidative stress, phenol biosynthesis, lignin deposition, and innate immunity, particularly in the suppressive soil. In tomato plants grown on steam-disinfected soils, total phenols and lignin decreased significantly. The inoculation of a mycorrhizal fungus partly rescued this response locally and systemically. Plants inoculated with the fungal pathogen showed reduced disease symptoms in the resistant genotype in both soils, but the susceptible genotype was partially protected from the pathogen only when grown on the suppressive soil. The 'state of alert' detected in tomatoes reveals novel mechanisms operating in plants in native soils and the soil microbiota appears to be one of the drivers of these plant responses.

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association.

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

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.

Gigaspora margarita with and without its endobacterium shows adaptive responses to oxidative stress.

Arbuscular mycorrhizal (AM) fungi experience oxidative stress during the plant-fungal interaction, due to endogenous reactive oxygen species (ROS) produced by fungal metabolism and exogenous ROS produced by plant cells. Here, we examine the responses to H2O2 in Gigaspora margarita, an AM fungus containing the endobacterial symbiont Candidatus Glomeribacter gigasporarum (CaGg). Previous studies revealed that G. margarita with its endobacterium produces more ATP and has higher respiratory activity than a cured line that lacks the endobacterium. This higher bioenergetic potential leads to higher production of ROS and to a higher ROS-detoxifying capacity, suggesting a direct or indirect role of the endobacterium in modulating fungal antioxidant responses. To test the hypothesis that the fungal-endobacterial symbiosis may enhance the fitness of the AM fungus in the presence of oxidative stress, we treated the fungus with a sublethal concentration of H2O2 and performed RNA-seq analysis. Our results demonstrate that (i) irrespective of the endobacterium presence, G. margarita faces oxidative stress by activating multiple metabolic processes (methionine oxidation, sulfur uptake, the pentose phosphate pathway, activation of ROS-scavenger genes); (ii) in the presence of its endobacterium, G. margarita upregulates some metabolic pathways, like chromatin status modifications and iron metabolism; and (iii) contrary to our hypothesis, the cured line responds to H2O2 by activating the transcription of specific ROS scavengers. We confirmed the RNA-seq findings by measuring the glutathione and ascorbate concentration, which was the same in both lines after H2O2 treatment. We conclude that both fungal lines may face oxidative stress, but they activate alternative strategies.

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis.

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.

Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi.

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

A critique of using epitaxial criterion to discriminate between protogenetic and syngenetic mineral inclusions in diamond.

Distinguishing syngenetic from protogenetic inclusions in natural diamonds is one of the most debated issues in diamond research. Were the minerals that now reside in inclusions in diamonds born before the diamond that hosts them (protogenesis)? Or did they grow simultaneously and by the same reaction (syngenesis)? Once previously published data on periclase [(Mg,Fe)O] and magnesiochromite (MgCr2O4) inclusions in diamond have been re-analysed, we show that the main arguments reported so far to support syngenesis between diamond and its mineral inclusions, definitely failed. Hence: (a) the epitaxial relationships between diamond and its mineral inclusion should no longer be used to support syngenesis, because only detecting an epitaxy does not tell us which was the nucleation substrate (there are evidences that in case of epitaxy, the inclusion acts as a nucleation substrate); (b) the morphology of the inclusion should no longer be used as well, as inclusions could be protogenetic regardless their shapes. Finally, we advance the hypothesis that the majority of inclusions in diamonds are protogenetic, e.g., they are constituent of rocks in which diamonds were formed and not products of reactions during diamond growth.

The genomes of Scedosporium between environmental challenges and opportunism.

Emerging fungal pathogens are a global challenge for humankind. Many efforts have been made to understand the mechanisms underlying pathogenicity in bacteria, and OMICs techniques are largely responsible for those advancements. By contrast, our limited understanding of opportunism and antifungal resistance is preventing us from identifying, limiting and interpreting the emergence of fungal pathogens. The genus Scedosporium (Microascaceae) includes fungi with high tolerance to environmental pollution, whilst some species can be considered major human pathogens, such as Scedosporium apiospermum and Scedosporium boydii. However, unlike other fungal pathogens, little is known about the genome evolution of these organisms. We sequenced two novel genomes of Scedosporium aurantiacum and Scedosporium minutisporum isolated from extreme, strongly anthropized environments. We compared all the available Scedosporium and Microascaceae genomes, that we systematically annotated and characterized ex novo in most cases. The genomes in this family were integrated in a Phylum-level comparison to infer the presence of putative, shared genomic traits in filamentous ascomycetes with pathogenic potential. The analysis included the genomes of 100 environmental and clinical fungi, revealing poor evolutionary convergence of putative pathogenicity traits. By contrast, several features in Microascaceae and Scedosporium were detected that might have a dual role in responding to environmental challenges and allowing colonization of the human body, including chitin, melanin and other cell wall related genes, proteases, glutaredoxins and magnesium transporters. We found these gene families to be impacted by expansions, orthologous transposon insertions, and point mutations. With RNA-seq, we demonstrated that most of these anciently impacted genomic features responded to the stress imposed by an antifungal compound (voriconazole) in the two environmental strains S. aurantiacum MUT6114 and S. minutisporum MUT6113. Therefore, the present genomics and transcriptomics investigation stands on the edge between stress resistance and pathogenic potential, to elucidate whether fungi were pre-adapted to infect humans. We highlight the strengths and limitations of genomics applied to opportunistic human pathogens, the multifactoriality of pathogenicity and resistance to drugs, and suggest a scenario where pressures other than anthropic contributed to forge filamentous human pathogens.

The Oral Microbiome for Geographic Origin: An Italian Study.

The human oral microbiome has primarily been studied in clinical settings and for medical purposes. More recently, oral microbial research has been incorporated into other areas of study. In forensics, research has aimed to exploit the variation in composition of the oral microbiome to answer forensic relevant topics, such as human identification and geographical provenience. Several studies have focused on the use of microbiome for continental, national, or ethnic origin evaluations. However, it is not clear how the microbiome varies between similar ethnic populations across different regions in a country. We report here a comparison of the oral microbiomes of individuals living in two regions of Italy - Lombardy and Piedmont. Oral samples were obtained by swabbing the donors' oral mucosa, and the V4 region of the 16S rRNA gene was sequenced from the extracted microbial DNA. Additionally, we compared the oral and the skin microbiome from a subset of these individuals, to provide an understanding of which anatomical region may provide more robust results that can be useful for forensic human identification. Initial analysis of the oral microbiota revealed the presence of a core oral microbiome, consisting of nine taxa shared across all oral samples, as well as unique donor characterising taxa in 31 out of 50 samples. We also identified a trend between the abundance of Proteobacteria and Bacteroidota and the smoking habits, and of Spirochaetota and Synergistota and the age of the enrolled participants. Whilst no significant differences were observed in the oral microbial diversity of individuals from Lombardy or Piedmont, we identified two bacterial families - Corynebacteriaceae and Actinomycetaceae - that showed abundance trends between the two regions. Comparative analysis of the skin and oral microbiota showed significant differences in the alpha (p = 0.0011) and beta (Pr(>F)= 9.999e-05) diversities. Analysis of skin and oral samples from the same donor further revealed that the skin microbiome contained more unique donor characterising taxa than the oral one. Overall, this study demonstrates that whilst the oral microbiome of individuals from the same country and of similar ethnicity are largely similar, there may be donor characterising taxa that might be useful for identification purposes. Furthermore, the bacterial signatures associated with certain lifestyles could provide useful information for investigative purposes. Finally, additional studies are required, the skin microbiome may be a better discriminant for human identification than the oral one.

Epitaxies of Ca sulfates on calcite. II. The main {010}, {001} and {100} forms of bassanite epi-deposited on the {10.4} substrate form of calcite.

2D and 3D epitaxies of the main {010}, {001} and {100} forms of deposited bassanite (CaSO4·0.5H2O) on {10.4} calcite (CaCO3) as a substrate are described to provide a theoretical crystallographic background for the replacement of calcite by bassanite both in nature and in the laboratory and by weathering linked to cultural heritage. First, epitaxy in the third dimension, perpendicular to the investigated interfaces, has been verified in order to establish whether adsorption/absorption can occur (as anomalous mixed crystals) at the bassanite/calcite epi-contacts. Secondly, and by applying the Hartman-Perdok method, 2D lattice coincidences have been obtained from the physical-geometric matches of bonds running in the common directions within the elementary slices facing the substrate/deposit interfaces. This research represents the second and more detailed part of a wider program extended to the epi-interactions between the following pairs: (i) {010}-gypsum/{10.4}-calcite (just published); (ii) bassanite/{10.4}-calcite (the present work); and (iii) anhydrite (CaSO4)/{10.4}-calcite (coming soon).

Microbial DNA in human nucleic acid extracts: Recoverability of the microbiome in DNA extracts stored frozen long-term and its potential and ethical implications for forensic investigation.

Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5-16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host's DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from "old" (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics.