Environmental Microbiome

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

Background Fungi in plant root systems are key to understanding the dynamics and functioning of terrestrial ecosystems. Whereas our knowledge of below-ground plant-fungus symbioses has derived mainly from studies of mycorrhizal and pathogenic fungi, recent studies have uncovered an unexpected diversity of ecological niches occupied by root-associated fungi. The fungal order Helotiales is conspicuous in this respect because this predominantly root-associated taxon encompasses a broad ecological spectrum, ranging from saprotrophic and pathogenic fungi to endophytic and mycorrhizal fungi with plant growth-promoting functions. In this study, we integrated a worldwide dataset of 1,996 Helotiales Species Hypotheses (i.e., species-level taxa tentatively defined based on DNA sequences) detected in roots of 362 plant genera representing 117 families and 47 orders, and systematically quantified the specificity of Helotiales-plant associations. Results The analysis of 37,688 unique occurrence records of Helotiales fungi across 1,940 global sampling locations revealed highly structured associations between Helotiales genera and plant lineages, exemplified by the strong specificity of Hyaloscypha, Oidiodendron, and Phialocephala for Pinales and that of Glutinomyces for Fagales, even after statistically controlling background biogeographic patterns. At the Species-Hypothesis-level analysis, major clades within the Helotiales phylogeny consistently exhibited high specificity for Pinaceae, whereas highly specific associations with other ectomycorrhizal, arbuscular mycorrhizal, and nonmycorrhizal plants were restricted to small terminal clades of the fungal phylogeny. Conclusions The integration of ecological specificity and phylogenetic analyses suggests the possibility that Helotiales fungi established close associations with Pinaceae early in their evolutionary history and subsequently underwent host shifts to diverse angiosperm lineages. Given the emerging evidence that Helotiales fungi play pivotal ecological and physiological roles in plant root systems, these statistical insights into Helotiales-plant associations provide a fundamental basis for understanding the evolutionary history and functioning of terrestrial ecosystems.

Mangroves persist under strong environmental constraints with support from microbes. Although microbial communities differ among mangrove compartments, their assembly mechanisms and compartment-specific functional signatures remain poorly resolved. Here, we combine peptide nucleic acid-clamping coupled with 16S rRNA gene amplicon sequencing and genome-resolved metagenomics to investigate the microbial communities associated with the leaves and roots of gray mangrove trees in the Red Sea. Our results suggest homogeneous selection and homogenizing dispersal as key processes of microbial community assembly in leaves. This phyllosphere hosts novel prokaryotic lineages, with some of its members probably synthesizing rhodopsins, plant polysaccharide-degrading enzymes, and gamma-aminobutyric acid, a metabolite that increase tolerance to salinity stress. Genomes affiliated with Desulfobacterales and Sedimenticolaceae taxa are abundant belowground, suggesting complementary sulfur- and nitrogen-cycling capacities similar as occur in other blue-carbon ecosystems. Comparison with root-derived genomes from cordgrass revealed a host-driven selection of Sedimenticolaceae species with convergent metabolic profiles. Overall, this study provides an integrative view about the microbial biology of gray mangrove trees, offering foundational insights into the diversity, ecology, and predictive functionality of their aboveground-belowground microbiomes.

Stratified benthic deposits offer crucial modern analogs for Precambrian biogeochemical dynamics, yet whether the vertical organization of deep sedimentary columns reflects depositional history, present-day geochemical conditions, or both remains poorly understood, particularly under extreme oligotrophy. Here, we present a depth-resolved exploratory survey of a continuous 34-cm sediment core partitioned into 11 discrete horizons (L1 to L11) from Poza Roja 2, an alkaline, sub-hypersaline pond in the Cuatro Cienegas Basin, Mexico. Integrating physical stratigraphy, organic carbon and nutrient stoichiometry, and 16S rRNA gene metabarcoding, we evaluated down-core microbial community structure alongside acute phosphorus scarcity. Molar C:Po and N:Po ratios exceeded canonical Redfield proportions (106:16:1) in every layer, by up to 48.5-fold and 87.8-fold, respectively, both peaking in layer L4. Unconstrained ordination (PCoA) and hierarchical clustering grouped the layers into three compositional zones: upper (L2-L4), intermediate (L5-L6), and basal (L8-L11), with L1 and L7 occupying transitional positions. Archaea accounted for 31.0% of prokaryotic reads, ranging from 8.3% (L5) to 43.7% (L9), and were dominated by SM1-50 (Thermoplasmatota) and Halobacteriota. The phototrophic heliobacterium Heliorestis peaked in the intermediate zone (47.6% of bacterial reads in L6). Constrained Redundancy Analysis models lacked statistical significance (P >= 0.12), highlighting the exploratory nature of a single core (n = 11). This pilot characterization provides a baseline for understanding deep biogenic archives under severe nutrient limitation.

Pyrroloquinoline quinone (PQQ) is known to promote plant growth, yet how the rhizosphere microbiome varies with PQQ concentration remains poorly understood. We applied metagenomic sequencing to characterize the taxonomic composition and functional potential of the tobacco rhizosphere microbiome across PQQ treatments at 0.5, 1 and 2 mol/L plus an untreated control. The rhizosphere microbiome was dominated by Pseudomonadota and Actinomycetota and differed among treatments in a concentration-associated, non-monotonic manner. The most pronounced differences were observed at 1 mol/L, where alpha diversity showed dimension-specific variation and beta-diversity analysis resolved the most distinct community profile. Taxonomic and functional profiles varied concurrently, with Streptomyces, Sphingomonas, Rhodanobacter and Lysobacter, together with functions related to biosynthesis, energy metabolism, colonization and environmental sensing, showing higher relative abundance at this concentration. No broad-spectrum antimicrobial effect was detected at the community level, and antibiotic resistance gene abundance showed no significant differences among treatments after correction for multiple testing. These findings provide a basis for further evaluation of PQQ in modulating the rhizosphere microbiome in continuous cropping systems.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

Background: Amplicon sequencing of the 16S rRNA gene is widely used to characterise microbial communities, but the performance of commonly used clustering and denoising pipelines has not been systematically evaluated for highly diverse environmental datasets. We therefore benchmarked four established clustering and denoising pipelines, VSEARCH cluster_size, UNOISE as implemented in VSEARCH, Swarm, and DADA2, using simulated microbial communities with known ground-truth compositions and evaluated how performance varied with species richness, abundance unevenness, sequencing depth, and minimum abundance threshold. Results: Differences among pipelines were small at low species richness but became greater as species richness increased. High average cluster purity did not necessarily correspond to accurate species-level recovery, as species could be split across multiple clusters or recovered incompletely. Among the pipelines, UNOISE may be preferable when high species representation and cluster purity are prioritised, while also showing strong reconstruction of relative abundance profiles, albeit with extensive species splitting and many unclustered reads. DADA2 showed similarly strong reconstruction of relative abundance profiles and less species splitting but represented fewer species and had lower cluster purity at higher richness. Swarm provided a balanced compromise between species representation and splitting, whereas cluster_size may be useful when limiting species splitting is a priority, despite weaker reconstruction of relative abundance profiles. Increasing the minimum abundance threshold reduced species splitting but also reduced the number of clusters and perfect clusters and, at higher thresholds, decreased concordance with ground-truth relative abundance profiles. These effects were more pronounced at lower sequencing depths. Analysis of seafloor sediment samples also showed threshold-dependent sequence loss, including the loss of sequences repeatedly detected across replicate samples. Conclusions: Pipeline performance in highly diverse 16S rRNA amplicon datasets varies with dataset characteristics, evaluation criteria, and parameter settings. Pipeline selection should therefore reflect dataset characteristics and the analytical objective rather than rely on a single measure of performance, and minimum abundance thresholds should be evaluated in relation to sequencing depth rather than applied as fixed defaults. These findings emphasise the importance of reconsidering established analytical practices and transparently reporting bioinformatic settings as 16S rRNA amplicon sequencing is increasingly applied to highly diverse environmental microbial communities.

Seed-associated microorganisms represent a promising resource for developing microbiome-based strategies against seed-borne pathogens. Here, we assembled synthetic microbial communities (SynComs) from cultivable members of the natural radish seed microbiota and evaluated their capacity to protect seedlings against the seed-transmitted fungal pathogen Alternaria brassicicola Abra43. Seven SynComs representing single- or multi-kingdom combinations of bacteria (B), filamentous fungi (F), and yeasts (Y) (B, F, Y, BF, BY, FY, and BFY) were inoculated onto radish seeds. We assessed their effects on disease incidence, seedling growth, microbial community assembly (ITS1 and gyrB gene metabarcoding) and pathogen colonization (metabarcoding and specific qPCR on dhn1 gene for A. brassicicola Abra43). Protection of radish seedlings against A. brassicicola Abra43 strongly depended on SynCom composition. The yeast fraction alone provided little protection, whereas the bacterial fraction partially reduced symptoms and maintained seedling growth despite substantial pathogen colonization, suggesting increased plant tolerance. SynComs containing filamentous fungi markedly reduced A. brassicicola Abra43 abundance, and the full multi-kingdom BFY SynCom provided the most consistent protection, combining reduced disease incidence, restored seedling growth, and low pathogen relative and absolute abundance. Inoculated strains, Alternaria alternata ABAI001 and Plectosphaerella sp. FL19 were enriched in protective SynComs and displayed antagonistic activity against A. brassicicola Abra43 in vitro. Together, these results show that seed-derived multi-kingdom communities can reshape early plant microbiota and that combining bacterial and fungal strains with complementary ecological functions can improve protection against a seed-borne fungal pathogen.

Anthropogenic climate change is driving increased temperature and altered precipitation patterns in tropical rainforests, yet the impact of severe drought on soil viral communities and their recovery capacity remains poorly understood. Soil viruses can exert strong controls on bacterial populations impacting microbially-mediated soil processes such as carbon cycling. At the same time, viruses are very sensitive to changes in the soil environment. We leverage meta-omics data from soils collected during a two-month drought and one-month recovery experiment carried in the Biosphere 2 Tropical Rainforest artificial biome. Our results indicate that soil viral community structure responds to small scale soil habitat heterogeneity more strongly than on overall changes in moisture. Additionally, the abundance of soil viruses was associated with a higher diversity of metabolites in the soil, suggesting viruses play a key role in soil biogeochemical processes. Altogether, our study suggests that soil viruses are an active part of tropical rainforest soil microbial communities and that is necessary to understand their response to drought.

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

Mangrove reforestation is expanding across the Caribbean, but restoration success is still judged almost exclusively by tree survival and canopy cover. Whether the sediment microbiome, which governs carbon burial and nutrient cycling, follows the recovery of the vegetation remains largely unknown. We combined 16S rRNA gene metabarcoding (V4, Illumina MiSeq) with CHNS elemental analysis, pH and salinity measurements to compare sediment prokaryotic communities along 0-100 cm profiles in a reference, a reforested (Rhizophora mangle plantings since 2019) and an altered mangrove within the Las Dunas de Las Calderas Natural Monument, Dominican Republic (39 cores pooled into 28 depth-resolved composite samples). Reference sediments contained 2.5-5.5-fold more organic carbon (median 7.5% vs 3.0% in altered and 1.3% in reforested sediments), had higher C/N ratios and were less alkaline (pH 8.2 vs 8.8). Reference communities were more diverse and even, and differed in composition from both disturbed stands (pairwise PERMANOVA, R^2 ~ 0.09, adjusted P = 0.0015), whereas reforested and altered communities were statistically indistinguishable (R^2 = 0.04, P = 0.25). Disturbed sediments were dominated by Cyanobacteriota (~41-45% of reads), which tracked high pH, whereas reference sediments were enriched in Chloroflexota, Thermodesulfobacteriota and Thermoproteota, lineages linked to anaerobic degradation of complex organic matter and sulfate reduction. Depth structured communities by separating surface from subsurface layers, and sediment organic carbon and nitrogen were the only significant constraints in distance-based redundancy analysis. These results indicate that, within a few years of the first plantings (2019), reforestation had not restored the carbon-rich, anaerobic sediment habitat of the reference forest. We propose sediment organic carbon and microbial community structure as complementary indicators for mangrove restoration monitoring in the Caribbean.

The mechanisms by which coarse woody debris (CWD) affects soil microbial diversity across different spatial scales and forest disturbance regimes remain poorly understood. This study investigated the impact of CWD from multiple tree species on forest floor bacterial and fungal communities under varying landscape-scale conditions, specifically comparing old-growth forests situated on Phaozems and Retisols with mass-windthrow areas located on loamy Retisols and sandy Podzols. We analyzed both chemical compound and metagenomic profiles of 63 CWD-associated soil samples collected from the Kaluzhskie Zaseki Nature Reserve (European part of Russia). High-throughput Illumina sequencing of the 16S rRNA gene and the ITS region was used to profile both bacterial and fungal communities, respectively. Scale-dependent diversity patterns and taxonomic accumulation were assessed using GLM, NMDS, dbRDA, and iNEXT frameworks. Our findings revealed a profound spatial decoupling in forest floor microbial communities, where a localized reduction in alpha-diversity under CWD drove a sharp increase in landscape-scale gamma-diversity. While standard soil physicochemical properties rigidly dictated baseline matrix richness, CWD operated as a powerful micro-environmental hotspot overriding these constraints. Deadwood inputs filtered out dominant matrix-associated taxa, creating highly specialized micro-refugia characterized by high compositional dispersion. This niche selection was particularly pronounced beneath Quercus logs on Phaozems and Retisols. Furthermore, post-windthrow microbial responses were heavily filtered by wood traits and baseline soil textures. On loamy Retisols within the windthrow mosaic, this breakaway dynamic was highly taxon-specific: while Acer and Fraxinus CWD triggered partitioned, site-specific hotspot dynamics yielding an exceptionally large pool of unique ASVs for bacteria, the distinct enrichment for fungal communities was primarily driven by Ulmus CWD and log-free windthrow areas. Ultimately, iNEXT accumulation profiles confirmed that deadwood does not merely multiply common soil taxa, but enriches the forest landscape by injecting critical beta-level heterogeneity into the soil matrix. These insights emphasize the role of diverse CWD retention in buffering forest microbiomes against large-scale environmental disturbances across heterogeneous soil landscapes.

As circular agricultural inputs are increasingly sought, feather hydrolysate (FH), derived from keratin-rich poultry waste, has emerged as a promising fertilizer and biostimulant. However, how FH shapes soil microbiome succession over time, and what functional potential this restructuring carries, remains poorly understood. Here, we used multi-marker high-throughput sequencing of 16S, 18S, and ITS rDNA to track soil microbial communities over 30 days following FH amendment, with sampling at days 0, 1, 4, 14, and 30. FH induced a rapid and reproducible microbial succession characterized by an early decline in alpha diversity and a pronounced day-1 bloom of copiotrophic taxa, as inferred from predicted rrn operon copy-number profiles. This initial response was accompanied by enrichment of bacterial genera previously associated with plant growth-promoting traits, including pathogen suppression, nutrient solubilization, siderophore production, phytohormone synthesis, and ACC deaminase activity. Although these groups gradually declined after the initial peak, several remained at significantly elevated abundances throughout the 30-day experimental period. In parallel, FH-treated soils showed a sustained reduction in the cumulative relative abundance of genera annotated as putative phytopathogens. By integrating temporal community profiling, rrn-based life-strategy inference, and literature-derived functional annotation, we show that FH acts as a strong ecological filter, shifting the soil microbiome toward copiotrophic and putatively plant-beneficial taxa. These findings provide a temporal framework for understanding FH-mediated microbiome restructuring and highlight the need for future studies linking inferred microbial functional potential to measured disease incidence, nutrient fluxes, and plant performance across diverse soil contexts.

Engineering bioenergy crops for enhanced biomass digestibility and high-value co-products is a promising approach to sustainable biofuel production. However, the impact of these engineered crops on the belowground microbiome remains poorly understood. In this study, we characterized the composition and functional responses of the belowground microbiome associated with transgenic sorghum lines: one with reduced cell wall crosslinking (AT10) and another with elevated protocatechuic acid (PCA) production (Qsub). The lines were grown under field conditions with four irrigation levels, and samples were collected twice during the growing season. We found that the two transgenic types drove a subtle but significant divergence in bacterial communities, both compositionally and functionally. Community-wide functional profiling showed that AT10 rhizospheres were consistently depleted in genes associated with mobile genetic elements, while Qsub rhizospheres were enriched in TonB-dependent transport and vanillate monooxygenase, an enzyme that funnels lignin-derived monomers toward PCA degradation, alongside reduced pectin- and cellulose-degradation genes. Genome-resolved analysis linked these community-level shifts to specific taxa: in Qsub, the enriched MAGs were taxonomically diverse, ranging from genome-reduced Patescibacteria with an episymbiotic lifestyle to taxa enriched in lignin-associated auxiliary activity enzymes. In AT10, the depleted Azospirillaceae MAGs carried unusually high mobile genetic element content, consistent with the community-wide pattern. Collectively, these findings offer new insights into how genetic modifications aimed at improving bioenergy traits shapes the composition and function of the rhizosphere microbiome, with potential downstream consequences for soil carbon cycling.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

Microbial community assembly on leaves (the phyllosphere) is influenced by deterministic, stochastic, and historical processes. However, the importance of historical processes such as priority effects, where the timing and order of colonization influence community assembly, has not been extensively investigated in field-based phyllosphere studies. We designed a field experiment in a temperate forest in Quebec where we inoculated five strains of Methylobacterium onto the leaves of red maple and American beech trees, either early or late during the growing season. We quantified microbial community structure throughout the season using metabarcoding of a Methylobacterium-targeting rpoB primer as well as a 16S rRNA primer targeting the entire bacterial community. We show that the effects of inoculation on bacterial communities depend on inoculation timing: early-season inoculation led to strain-specific shifts in community composition, while late-season inoculation had no significant effect. Although the effect of early-season inoculation weakened over time, early- and late-season inoculation still led to different community compositions at the end of the growing season. Our study demonstrates that priority effects contribute to the assembly of phyllosphere bacterial communities under field conditions, with arrival timing and strain identity shaping community trajectories. Considering historical processes will thus be crucial for successful microbiome management.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

Cyclosporiasis is a foodborne illness caused by the parasite Cyclospora cayetanensis. Clinical surveillance often lags infection by weeks because diagnosis depends on a clinician ordering a test that is not part of routine parasite panels. Wastewater metagenomic sequencing does not depend on care seeking, which may be useful for early detection and community monitoring. We designed 1,670 31-mers from C. cayetanensis mature ribosomal RNA and retained 1,464 without an exact non-target match in the 28 July 2025 NCBI core_nt release. By analyzing 293 public wastewater metagenomes, we selected a threshold of 24 distinct diagnostic 31-mers on one read. Applying that threshold to 2,333 public runs from BioProject PRJNA1247874 yielded putative C. cayetanensis reads in 81 runs, or 3.5%, and in 24 of the 30 longitudinally sampled sewersheds. Detections rose through the summer in two consecutive years, matching the known seasonality of cyclosporiasis. The most parsimonious explanation is that these reads come from parasites shed into wastewater, although undescribed Cyclospora species absent from reference databases cannot be excluded. The same approach can be applied to other pathogens in existing wastewater sequencing data.

Carbon farming practices are increasingly adopted as part of regenerative agriculture to enhance soil carbon sequestration and improve agricultural sustainability. However, their effects on the phyllosphere microbiome, and how these interact with field biodiversity, remain poorly understood. We investigated how field biodiversity and carbon farming practices (cover crops, all-in mixes, adaptive grazing and ley mixture) influence leaf microbial communities across 19 Finnish farms using ITS and 16S amplicon sequencing. Foliar fungal - but not bacterial - diversity increased with plant species richness. Carbon farming altered both fungal and bacterial community composition, but only fungal community was positively associated with disease load. Network analysis identified three candidate keystone taxa: Alternaria sp. (FASV 34) and Sporobolomyces roseus (FASV 2070), which were mutually exclusive, and Methylobacterium sp. (BASV 75), which co-occurred with FASV 2070. Together, these findings demonstrate that different carbon farming practices shape distinct leaf microbiomes cover crop systems tending to favor potential fungal pathogens, whereas ley mixture and adaptive grazing strategies promote microbial taxa associated with healthier phyllosphere communities. Our study underscores the importance of understanding the complex microbial interactions within leaf ecosystems to inform sustainable agricultural practices.

Functional profiling of anaerobic digestion microbiomes is routinely performed against KEGG or MetaCyc, whose paid commercial licences block fee-for-service analysis for biogas operators, the setting where the results have the most immediate operational value. We present digestome, a curated marker-gene panel for anaerobic digestion built exclusively on sources that are free for commercial use (NCBIfam, public domain; Pfam, CC0; Rhea, CC BY), together with a scorer that reports pathway completeness, branch capability and, for hydrolysis, whether the enzyme is built for export. Benchmarked against 1,401 metagenome-assembled genomes from 134 anaerobic digesters, the panel assigned no methanogenesis route to any of 1,361 non-methanogens, and every acetoclastic call fell within Methanosarcina or Methanothrix. The result held for 3,043 species representatives from the Genome Taxonomy Database, spanning 203 phyla, which are built differently from binned metagenomes: the only background genomes given a route were two archaea carrying mcrA, and none of the 90 anaerobic methane and alkane oxidisers was given one. Specificity against non-methanogens does not show that a methanogen gets the right route: with a family-level check, 59 of 156 acetoclastic calls in that sample fell on methylotrophic genera that do not use acetate, and a lineage policy removed them, with 14 more left unconfirmed in unnamed genera. Testing whether a catalytic domain shares a polypeptide with an export module reduced the genomes credited with cellulolytic capacity from 597 to 50. A causal graph of the measurement shows that a profile reports the community together with three measurement choices (sampling, DNA extraction and sequencing depth, and reference genomes), which fixes what one profile can attribute and what a prospective validation would have to record.