Microbial Ecology

Ectomycorrhizal (EcM) fungi are well-recognized symbionts impacting tree health and ecosystem functioning globally, yet understanding of their timing of proliferation in soils across seasons and years remains limited. We analyzed monthly patterns of EcM fungal abundance and community structure over two years in five temperate monodominant forest plots via quantitative PCR and Illumina sequencing. We found that the phenological dynamics of EcM fungi differed significantly by host tree leaf habit, fungal exploration type, fungal genus, and soil moisture. Overall, total EcM fungal abundances based on qPCR consistently peaked in autumn, and were more dynamic in evergreen than deciduous plots, supporting ideas of surplus carbon and asymmetric above-belowground dynamics. Longer-distance exploration types peaked earlier and were more stable than shorter-distance types, suggesting an independent and supportive role in releasing spring nutrients. About half of 20 focal taxa consistently peaked in either autumn, summer, or spring, while others were either host- and/or year-dependent. Our findings highlight that phenology is a key EcM fungal trait best explained by both host and fungal contributions, and future studies across biomes should consider seasonal shifts and sampling to elucidate phenological traits. Summary- The timing of belowground production and seasonal community dynamics remain poorly understood for ectomycorrhizal (EcM) fungi. - We collected soils monthly for two years from five temperate monodominant forest plots. - Fungal production peaked in autumn, shorter-distance and evergreen-associated spanned wider ranges, and half of focal fungal genera showed seasonal preference, emphasizing autumn surplus carbon and spring nutrients from long-distance types. - Future studies should consider seasonal shifts when sampling EcM fungal communities, and forest carbon models should include asymmetric above-belowground phenology. Translated Summary (Spanish)- La fenologia de la produccion y composicion de comunidades de hongos ectomicorrizicos (EcM) es poco estudiada. - Recolectamos suelos mensualmente por dos anos de cinco parcelas mono-dominantes templados. - Produccion maxima de hongos ocurrio en otono, hongos asociados con arboles siempreverdes y de exploracion de corta-distancia observaron rangos mas amplios, y la mitad de generos de hongos focales observaron preferencia estacional, enfatizando extra carbono en otono y nutrientes en primavera de tipos larga-distancia. - Estudios deben considerar cambios estacionales para el muestreo de hongos EcM, y modelos de carbono deben incluir fenologia asimetrica entre hojas y hongos. Plain language summaryEctomycorrhizal fungi are critical for the global carbon cycle, but their seasonal and inter-annual growth patterns remain unclear. We sample soil DNA monthly over two years across five different monodominant temperate forest stands. We find an overall belowground peak in autumn, with significantly later growth under wetter conditions, more dynamism with evergreen trees, and distinct spring growth by longer-distance fungi.

Microbial communities are central to the biogeochemical cycling of nutrients, critically shaping ecosystem functioning and influencing climate change mitigation. Mangrove ecosystems are among the most important global carbon sinks that enable large amounts of carbon to be sequestered and stored. However, gaps persist in understanding the fundamental aspects of microbial-driven carbon cycling in these environments. This research explores the microbial taxonomic and functional diversity related to carbon cycling in selected tropical mangrove sediments across various locations and depths. Sequencing data analyses based on the 16S rRNA gene revealed distinct microbial community composition but conserved predicted functions across the different mangrove locations. Depth was a strong influence on the functional composition, with carbon-related pathways and metabolic strategies differing between top and bottom sediments. Putative functional gene abundance analyses revealed that carbon fixation processes were among the top carbon-related pathways, suggesting the key role of mangrove microbial communities in sustaining long-term carbon storage. Within these communities, Desulfobacterota appeared as a primary contributor to carbon fixation, while Chloroflexota played a significant role in carbon metabolism and methane cycling. Co-occurrence network analyses also revealed that these microbial groups were among the keystone taxa in mangrove sediments. Our study adds on to the body of knowledge on the mangrove microbiome and their carbon metabolic processes, which helps to improve strategies for managing and leveraging these vital carbon sinks.

The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While several studies have characterised the composition and roles of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used 16S rRNA gene sequencing to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Analysis of 239 samples revealed a set of dominant bacterial genera commonly associated with other fish species, including Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter, that constituted a core skin microbiota across lochs. Microbiome composition was primarily shaped by environmental variables, particularly habitat, water pH, conductivity, and metal concentrations, with pH emerging as a key driver of community structure. Host sex also influenced microbiome variation, with several taxa differing in relative abundance between males and females. Alpha-diversity was higher among stickleback fish from lochs with a neutral pH compared with those from alkaline and acidic environments. Differential abundance analyses identified 27 and 24 amplicon sequence variants (ASVs), respectfully, associated with variations in pH and host sex, including members of Psychrobacter, Sphingobacterium, Carnobacterium, Chryseobacterium, and Arthrobacter, highlighting the combined influence of environmental and host factors on microbiome composition in wild fish populations in freshwater environments.

The evolutionary divergence between Henophidia (non-venomous) and Caenophidia (venomous) snakes has produced distinct cranial morphologies, digestive strategies, and presence of specialised venom systems in Caenophidia, yet the extent to which these long-standing diverging trajectories have shaped cloacal microbiota assembly remains poorly understood. We characterised cloacal microbiota in 70 captive snakes (52 Caenophidia, 18 Henophidia) by 16S rRNA amplicon sequencing. Beta diversity was tested by PERMANOVA, differential abundance by ANCOM-BC2, community types by Dirichlet Multinomial Mixture modelling (DMM), and microbial interactions by SparCC co-occurrence networks. Predicted functional potential (PICRUSt2) was analysed by ALDEx2 differential abundance testing and elastic net feature selection. Henophidia exhibited significantly higher bacterial richness and greater compositional variability than Caenophidia. Community composition showed clade-associated differences (PERMANOVA) and partitioned into two distinct DMM community types. The Henophidia network was 11.9-fold denser and more modular, with Burkholderiaceae as a keystone hub, whereas the Caenophidia network was sparse. Henophidia showed predicted enrichment in C1 metabolic pathways (ethylmalonyl-CoA, formaldehyde assimilation I, glycine betaine degradation I, methylaspartate cycle), aromatic compound catabolism, and nitrogen recycling, whilst Caenophidia showed enrichment in allantoin and glucuronate degradation. This multi-method analysis suggests Burkholderiaceae as a candidate keystone taxon in Henophidia and indicates that phylogenetic clade is a major contributor to cloacal microbiota structure. The lower richness in Caenophidia raises a testable hypothesis that broad-spectrum antimicrobial activity of their venom components may selectively filter susceptible microbial lineages, motivating future shotgun metagenomic studies in wild populations of snakes.

BackgroundAdult vestimentiferan tubeworms inhabiting hydrothermal vents and cold seeps lack a mouth and anus and rely entirely on organic matter produced by sulfur -oxidizing autotrophic bacterial symbionts in their trophosomes. These symbionts, which predominantly belong to the genus Proteobacteria, are acquired horizontally from the environment. However, the effects of rearing conditions that differ from natural habitats on the microbiome composition or abundance of these bacteria remain unclear. MethodsWe conducted a metagenomic analysis of Lamellibrachia satsuma reared in an aquarium under sulfide-supplemented and sulfide-free conditions. ResultsImmediately after collection, the microbiome was dominated by known symbionts within {gamma}-Proteobacteria, exhibiting low species diversity. After 6 months of rearing, the abundance of these symbionts significantly decreased under both conditions, whereas overall bacterial diversity increased. In particular, -Proteobacteria became more abundant under sulfide-supplemented conditions, while {delta}-Proteobacteria predominated in the absence of sulfide. Despite these changes, symbionts were not entirely lost, and the hosts survived for 6 months, likely due to their low metabolic rate. These findings suggest that the microbiome of L. satsuma can respond flexibly to changes in the rearing environment. They also indicate that the hosts metabolism can be maintained even with a smaller quantity of symbiotic bacteria.

Short-read amplicon sequencing is widely used for fungal surveys but can limit taxonomic resolution. Long-read sequencing enables recovery of the full internal transcribed spacer (ITS) region and may improve ecological and taxonomic inference. Here, we conducted a paired comparison of Illumina ITS2 and PacBio HiFi full-length ITS sequencing using identical DNA extracts from built-environmental air and surface samples (n = 68) collected across homes, a dormitory, and laboratories. Both datasets were taxonomically assigned using the same algorithm and reference database. We performed paired statistics, in-silico ITS2 trimming of long-read sequences, and cross-platform mapping at multiple identity thresholds. Full-length ITS provided higher taxonomic resolution, assigning a greater fraction of ASVs at the family (98% vs. 88%) and species (42% vs. 32%) ranks than ITS2 (paired Wilcoxon q = 0.002). Alpha-diversity comparisons showed similar Shannon diversity across pipelines, whereas richness metrics were consistently higher for full-length ITS. Beta-diversity analyses indicated broadly comparable community-level patterns, although full-length ITS revealed stronger sample-type- and location-associated structure (PERMANOVA R{superscript 2} [≥] 0.06, p = 0.0001). In-silico ITS2 trimming reduced these differences, indicating that amplicon length is a major contributor to enhanced taxonomic resolution and ecological inference. Cross-platform mapping further showed extensive one-to-many relationships between ITS2 and full-length ITS ASVs, consistent with increased sequence resolution in long-read data. Together, these results show that ITS2 sequencing provides robust community-level profiling, while full-length ITS enables improved richness estimates and finer ecological and taxonomic resolution. This paired, bias-aware framework provides a practical template for selecting fungal amplicon sequencing strategies in built-environment mycobiome studies. ImportanceFungal communities in built environments influence indoor air quality and human exposure, yet their characterization depends strongly on sequencing strategy. This study provides a controlled, paired comparison of short-read ITS2 and long-read full-length ITS sequencing, showing that differences in amplicon length substantially contribute to variation in taxonomic resolution and ecological inference. While both approaches yield comparable community-level patterns, full-length ITS improves richness estimates, species-level assignment, and environmental discrimination by resolving sequence variation collapsed in ITS2 surveys. By integrating paired diversity analyses, in-silico ITS2 trimming, and cross-platform ASV mapping, this work offers a bias-aware framework for evaluating fungal amplicon pipelines. Importantly, improved species-level resolution enables functional interpretation of indoor fungi, for example the identification of taxa associated with pathogenic traits, allergen production, or toxin synthesis, supporting the development of more informative exposure metrics and targeted assays relevant to human health in built environments.

Corals harbor a diverse bacterial community that facilitates adaptation and sustains their health. In coral holobiont research, culture-independent approaches have transformed the existing paradigm. Molecular techniques, such as metabarcoding, revealed a high diversity of previously unrecognized bacterial symbionts. Coral microbiota characterization has relied on these techniques over the last decade, but relying solely on them does not provide a detailed understanding of the dynamics of the coral holobiont complex. Returning to classic microbiological methods and in vitro experimentation can yield novel insights into symbiont roles, physiology, and interactions within the holobiont. Under this premise, we aimed to isolate and culture bacteria from four Mediterranean corals. The recovery of 84 pure bacterial isolates and their initial classification based on the 16S rRNA gene revealed substantial diversity among symbionts amenable to culture. Several isolates represent novel species within relevant genera, such as Vibrio, underscoring the value of culture-based studies. All cultures were cryopreserved to guarantee long-term accessibility for future projects. This represents a key step towards describing the roles of bacteria within the coral holobiont, as cultures enable in-depth morphological and physiological characterization of the symbionts and experimental ecology studies.

Near-shore coral reefs in southern Guam (Mariana Islands) experience severe sedimentation, in particular during the wet season when rainfall and erosion are high. We sampled fragments of the reef-forming coral Porites lobata from opposite ends of a sedimentation gradient in Fouha Bay, southern Guam, during dry and wet seasons. Using DNA metabarcoding, we characterized the diversity and composition of P. lobata-associated Symbiodiniaceae and bacterial microbiome communities. As in many species of Porites, Symbiodiniaceae communities of P. lobata were dominated by variants of Cladocopium C15 with sites showing differences in Symbiodiniaceae communities attributable to variation in these Cladocopium C15 variants. Bacterial microbiomes of P. lobata were dominated by Endozoicomonadaceae, a family of putative coral bacterial endosymbionts involved in nutrient cycling. Site and seasonal differences in bacterial diversity and community composition were apparent. In close proximity to the mouth of the river draining into Fouha Bay, bacterial diversity was highest during the wet season when sedimentation is generally severe. Microbiome reorganization in response to sedimentation may explain this result, but we also found overrepresentation of bacteria associated with terrestrial origin close to the river mouth and/or during the wet season. Together these patterns highlight that coral Symbiodiniaceae and bacterial communities are both spatially and temporally structured in this disturbed system. IMPORTANCEThis study provides a time series dataset of coral-associated microorganisms, including dinoflagellate algae and bacteria, from a tropical bay impacted by sedimentation that results from upstream erosion of disturbed soils. Characterizing temporal patterns of coral-associated microbes provides insights into the dynamic nature of these communities. While microbiome variability across sites and seasons may be a result of acclimatization to different environmental conditions, we identified bacterial groups of putative terrestrial origin in sampled coral microbiomes that may have been exported from eroded soils to the near-shore reef. Considering that disturbed soils act as hotspots for the proliferation of potentially harmful substances, such as antimicrobial resistance genes, understanding microbial community connections at the marine-freshwater-terrestrial interface is an important step toward evaluating environmental impacts across connected ecosystems from ridge to reef.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

O_LIA productivity-driven higher nutrient demand of trees in diverse mixtures is frequently reported. Yet, it remains unclear how tree diversity influences microorganisms-plants interactions, in which microbes facilitate tree nutrient acquisition in exchange for carbon (C) to meet the resource demand of both. C_LIO_LIUsing a long-term tree diversity experiment in the subtropics, we assessed microbial investment in C-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes in litter and mineral soil, testing the effects of tree species richness and mycorrhizal type (arbuscular (AM)- vs. ectomycorrhizal (EcM)-associated tree species). C_LIO_LIWith increasing tree species richness, microbial investment in C acquisition decreased, while investment in N and/or P acquisition increased in litter and in mineral soil. In mineral soil of AM-associated tree mixtures, ecoenzymatic stoichiometry revealed a shift from microbial investment in C toward P acquisition as tree species richness increased. C_LIO_LIOur findings suggest that tree diversity strengthens microbe-tree interactions in terms of C-for-nutrient exchange. This highlights the key role of soil microorganisms, particularly in AM symbiosis, shaping tree diversity-biogeochemical feedbacks. C_LI

Terrestrial environmental DNA (eDNA) approaches are rapidly expanding, yet robust, field-ready substrates for detecting insect DNA remain limited in forest ecosystems. Tree sap is a localized microhabitat that attracts diverse insects and may provide a useful substrate for surface eDNA sampling, but its potential for insect monitoring has rarely been evaluated. Here, we present a pilot proof-of-concept study testing naturally exuding tree sap and sap-mimicking traps as terrestrial eDNA substrates. We collected swab samples from sap and trap surfaces at two forest sites in Japan (Fujisawa and Minamisanriku) and performed metabarcoding using COI and an arthropod-focused 16S marker (gInsect). Reads were processed into amplicon sequence variants and assigned by BLAST top hits against NCBI nt, with high-confidence detections defined at identity [≥]98%. Across sites, sap and trap swabs yielded multiple high-confidence insect detections spanning several orders, including sap-associated stag beetles (Dorcus spp.). Overlap with contemporaneous conventional monitoring was limited, suggesting that sap-surface eDNA and conventional surveys capture partly different components of sap-associated insect assemblages. In a targeted 2024 spot survey, actively fermenting sap yielded multiple insect eDNA detections, whereas inactive, non-fermented sap yielded no high-confidence insect detections. Although limited by small sample size and the absence of dedicated process controls, these findings support the feasibility of tree sap as a localized terrestrial eDNA substrate and provide a basis for future replicated studies of sap-associated insect monitoring.

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

There is disagreement on whether secondary endosymbionts are found in the major cereal pest aphid, Rhopalosiphum padi. Some papers report a diversity of secondary bacterial endosymbionts while others have failed to find evidence of these bacteria in this species. Here we revisit this issue by summarizing the relevant literature and through additional sampling of the species in Australia, China and Denmark using a combination of molecular approaches. We find a general absence of secondary endosymbionts beyond the obligate endosymbiont Hamiltonella defensa in R. padi. While the inconsistency in survey results may reflect rapid changes in endosymbiont turnover in populations and/or the impact of ecological factors such as host plant type on endosymbiont diversity, we are concerned that technical issues may be at least partly responsible for inconsistencies in the literature. This leads us to emphasize the importance of multiple sources of evidence required to establish and characterize endosymbiont infections, including PCR and qPCR assays, DNA Sanger sequencing and 16SrRNA gene metabarcoding. We note that several major aphid pests show a low incidence of secondary endosymbionts which raises issues about the importance of these endosymbionts in aphids that constitute pests, even though endosymbionts can in some cases increase host fitness and therefore pest impact.

Riverine ecosystems particularly in industrialized environment are subjected to chronic press disturbances, resulting from the decadal release of synthetic organic compounds and other xenobiotics. While indigenous microbial communities are highly sensitive to such stressors, the resulting metabolic restructuring and functional reshaping of the microbiome, driven by these long-term anthropogenic pressures remains poorly characterized. In this study, a microbial ecology of Bhadar River flowing across the Jetpur Industrial Estate, (Jetpur) were studied. Using a cross-sectional comparative approach, soil/sediment samples were collected from the diverse polluted and non-polluted sites from the estate. The taxonomic profiling using 16S rRNA gene amplicon sequencing, taxo-phenomic shifts (through metaphenomics) was studied, while the functional potential of metabolic pathways was validated using high-resolution shot-gun metagenomic study. Due to prolong pollution, the samples were rich in sulphur (9809 to 12391 mg/L), where polluted samples were having elevated COD (2432 to 4150 mg/L) as well as BOD (1000 to 1420 mg/L) values, along with the presence of heavy metals (e.g., Fe, Mg). Results revealed a distinct taxonomic shift at both the bacterial and archaeal levels. In non-polluted sites Proteobacteria (33 to 57%) dominated along with Acidobacteria and Actinobacteria, with diverse genera like Alcaligenes and Serratia. Whereas, polluted sites exhibited marked increase in Bacteroidetes (13 to 29%), Firmicutes, and Synergistetes and genera like Alkalitalea, Mesotoga and Desulfomicrobium, reflecting anaerobic, fermentative, and sulfate-reducing phenotypes. The archaeal communities at polluted sites were dominated by Euryarchaeota (78 to 99%), specifically methanogenic genera of Methanosaeta and Methanocalculus, contrasting with the Methanomassiliicoccus dominance in non-polluted areas. The alpha-diversity was marginally higher in polluted sites (Shannon: 4.11 to 4.81 vs. 3.81 to 5.39 (non-polluted)), but beta-diversity underscored clear separation (94% variance explained by pollution). The shot-gun metagenomic analysis indicated a substantial enhancement in anaerobic metabolic capacities within the polluted microbiome, primarily in sulphur respiration (dissimilatory sulfate reduction), methanogenesis (elucidating biogenic pathways), along with nitrogen cycling (identifying key denitrification and ammonification genes). The polluted microbiome have developed the potential to metabolise/degrade complex aromatic compounds (pcaK for benzoate/protocatechuate transport) and heavy metal resistance. The strong positive co-occurrences among anaerobic phyla (Thermotogae, Synergistetes, Bacteroidetes) in polluted sites was established, indicating syntrophic interactions for xenobiotic metabolism. These findings provide a theoretical ecological model for perturbed industrial ecosystems, emphasizing the role of habitat selection in shaping microbial functional diversity and demonstrate the remarkable adaptation of autochthonous communities to persistent press disturbances.

Industrialization has been identified as the single biggest factor driving global microbiome diversity. While many studies examining gut microbiomes attribute these shifts to dietary increases in fat and reductions in protein, oral microbiome responses to industrialization remains debated. The oral microbiome is more resilient due to long-standing coevolution with host tissues and biofilm stability. However, limited geographic and historical representation has constrained our understanding of how these transitions unfolded globally in the oral microbiomes. Here, we investigate oral microbiome variation in Batwa rainforest hunter-gatherers and neighboring Bakiga subsistence farmers from southwestern Uganda, comparing them with publicly available data from Tanzanian, Venezuelan, and industrialized populations from North America, Europe, and Australia. Using 16S rRNA gene sequencing, we characterized salivary microbiota and evaluated differences in local and global diversity, composition, and differential abundance. Ugandan populations contained significant compositional differences but similar levels of diversity, suggesting that shared environments and dietary overlap may shape microbial assemblages despite distinct cultural histories. Globally, strong continental and industrialization effects were observed in the oral microbiome, with all industrial populations clustering separately from people living in other locations. African populations also clustered separately from non-African groups. Oral microbiome diversity was highest in Ugandan individuals and lowest in industrialized populations, mirroring patterns previously observed in the gut microbiome. Together, these findings demonstrate that both geography and subsistence strategy structure global oral microbiome variation. They also clarify the position that oral microbial communities record biocultural transitions and highlight the need to better understand the industrial mechanisms that shape microbial diversity in the oral cavity.

Metabarcoding is a powerful tool for biodiversity comparisons, where standard-size DNA barcodes (>500 bases) offer better taxonomic resolution than shorter ones. Still, the choice of sequencing platforms and bioinformatics pipelines may strongly affect inferred diversity due to various technical biases. We assessed the relative performance of Illumina MiSeq i100 (2x500 paired-end), PacBio Revio and Oxford Nanopore MinION sequencing and bioinformatics pipelines, using full-length ITS amplicon sequencing datasets from a 103-species mock community and 45 composite soil samples. Despite numerous low-quality reads, PacBio yielded the lowest overall error rate and highest number of taxa. Illumina revealed the highest proportion of chimeric and index-switched reads, along with a strong bias towards shorter amplicons. MinION data analysed using PRONAME and Minovar - a bioinformatics pipeline presented here - had the largest proportion of low-quality data, and rare taxa were lost during data filtering and read polishing steps. Although Minovar enabled amplicon sequence variant (ASV) level precision for common taxa, we recommend clustering ASVs into OTUs. For PacBio, standard filtering approaches outperformed the ASV approach because they retained rare taxa. For Illumina, a stringent ASV approach or removal of rare OTUs would limit artefacts. Across all platforms, excess PCR cycles promoted chimeric and low-quality reads and lost quantitativity in biodiversity assessments. With moderate differences in effect sizes, all analytical approaches supported the conclusion that sampling design determines how we see soil biodiversity responses to land use. For biodiversity surveys based on the full-length ITS metabarcoding, we recommend using PacBio sequencing with standard, non-ASV pipelines.

Plant growth promoting microbes enhance developmental progression of the host by influencing its nutrient availability or by deploying secondary metabolites responsible for manipulating the hormonal crosstalk. Microbacterium bengalense sp. nov. GB16_1_BI (Accession number: SRX9280401), a newly identified ammonium releasing Actinomycetota, could enhance plant growth by manipulating rhizosphere bacteria. Amplicon sequencing of the 16S rRNA V3-V4 region from the rhizosphere of the black rice (Chakhao Poireiton) showed that GB16_1_BI could inhibit most bacteria. However, GB16_1_BI inoculation encouraged the growth of rare bacteria specific to waterlogged rice rhizosphere. Analysis of the OTUs using PICRUSt2 (Phylogenetic investigation of communities by reconstruction of unobserved states) showed increased abundance in the marker genes for nitrogen cycling (nifH, nrfA and nrt) but not for nifD or nifK which was also reflected in the ANOSIM analysis in the OTUs of the N-fixing bacteria. Marker genes for methane metabolism (comA, comB, cofG and cofH) were also more abundant in the inoculated plants than the control; however, ANOSIM studies did not support this observation in the OTUs of methane cycling bacteria. Both Methylosinus and Methylocystis, the two most abundant methanotrophic OTUs, are also known to be nitrogen fixers. Hence, GB16_1_BI could influence plant growth predominantly by manipulating nitrogen cycling microbes. The genome sequence as well as untargeted metabolome analyses of GB16_1_BI showed abundance of secondary metabolites with probable antimicrobial activity. GB16_1_BI could utilize varied carbohydrates and amino acid as energy source and form persister-like cells may help it to survive in the soil in absence of the host plant.

Agricultural fields in the Mezquital Valley, Mexico, were irrigated with untreated wastewater over several decades. Following the construction of a wastewater treatment plant (WWTP) in Atotonilco de Tula, WWTP effluent is used for irrigation. To evaluate the effects of changed irrigation, a soil incubation experiment was performed. Soils of the Mezquital Valley long-term irrigated with untreated wastewater were irrigated with WWTP influent or effluent, both unspiked and spiked with antibiotics and biocidal compounds and incubated four weeks. We investigated the effects of shifted irrigation on the abundance of cultivable total heterotrophic and resistant bacteria (RB). Additionally, RB were cultivated from Coriandrum sativum (cilantro) sown in soil of the incubation experiment. While wastewater treatment significantly reduced the bacterial abundance in effluent, spiking increased RB abundance in both wastewater types including ciprofloxacin (CIP) RB. Before wastewater addition, all soils contained cultivable RB. Irrigation increased the relative abundance of RB cultivated on Mueller Hinton (MH) agar in Leptosols and Phaeozems, compared to soils prior to wastewater addition irrespective of the water type, but not in Vertisols, suggesting the soil type rather than water qualities influenced the RB abundance. Diverse CIP RB were cultivated including strains of 14 genera of three phyla. Among those, Achromobacter spp. strains related to potentially pathogenic A. spanius originating from soil were abundant in both leaves and roots of cilantro. Our results showed that the implementation of wastewater treatment does not reduce the abundance of cultivable RB in Mezquital Valley soils and cilantro plants. Health risk associated monitoring should include long-term persistent RB colonizing plants cultivated in wastewater irrigated soils.

Microbial colonization is a major cause of deterioration in paintings, leading to discoloration, pigment degradation, and loss of structural integrity. While biodeterioration of artworks has been studied in temperate climates, tropical environments remain underexplored despite their high humidity and temperature, which promote microbial growth. This study assessed the microbiological deterioration of two eighteenth-century oil paintings, La Muerte de San Jose and Virgen de Guadalupe, located in Orosis Colonial Church and Religious Art Museum, Costa Rica. Microorganisms were isolated and identified using VITEK(R) 2, microscopy, and MALDI-ToF analysis, and their biofilm-forming capacity was evaluated. Additionally, the antimicrobial activity of six essential oil components was tested using direct and indirect contact assays. Twenty-three bacterial species and fifteen fungal genera were identified, with Bacillus, Staphylococcus, Cladosporium, and Aspergillus among the most common. Notably, La Virgen de Guadalupe displayed the highest microbial diversity, reflected in a high Shannon index, indicative of a more complex microbial community. Several isolates displayed strong biofilm formation, particularly Bacillus subtilis/amyloliquefaciens/vallismortis and Staphylococcus saprophyticus. Linalool exhibited the strongest inhibitory activity, achieving complete bacterial growth inhibition in non-contact assays. Environmental monitoring revealed persistently elevated relative humidity and CO2 levels during the study period. Together, these results reveal the complex microbial ecology of tropical heritage paintings and demonstrate that volatile essential oil components can serve as candidates for low-impact antimicrobial strategies in preventive conservation. ImportanceUnderstanding the microbiological deterioration of cultural heritage in tropical environments is crucial for designing sustainable conservation strategies. While microbial colonization of artworks has been widely studied in temperate regions, data from tropical climates remain limited despite inherently favorable conditions for microbial proliferation. This study integrates microbiological, environmental, and physicochemical analyses to characterize microbial communities colonizing eighteenth-century oil paintings in Orosi, Costa Rica. By combining microbial identification, biofilm quantification, and essential oil biocide testing, it bridges applied microbiology and cultural heritage conservation. The finding that volatile components such as linalool inhibit biofilm-forming bacteria without direct contact highlights their potential as eco-friendly, noninvasive antimicrobial alternatives to conventional biocides. These results expand the understanding of biodeterioration dynamics under tropical conditions and offer a practical framework for developing sustainable, evidence-based conservation protocols that protect both heritage materials and the environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/723565v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@16cd608org.highwire.dtl.DTLVardef@57aa00org.highwire.dtl.DTLVardef@159fcbeorg.highwire.dtl.DTLVardef@e0363b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0.C_FLOATNO Artistic visualization of the geographical context of the studied artworks and the multidisciplinary analytical approaches applied, highlighting the diversity of microorganisms identified (illustration by Keylin Urena-Alvarado). C_FIG

Northern peatlands store large carbon stocks but are sensitive to disturbance. Hydrology, vegetation, herbivory and snow conditions may affect the soil microorganisms driving methane (CH) and nitrous oxide (N2O) cycling. We investigated how reindeer exclusion and snow depth (increased and reduced relative to ambient) manipulations (ongoing for three seasons) influenced archaeal and bacterial communities in a boreal rich fen. Metagenomic (MG) and metatranscriptomic (MT) sequencing were combined with pore-water chemistry and CH flux measurements to link the microbiome to ecosystem processes. Microbial communities differed between outside and inside the exclosure. However, these patterns primarily reflected underlying hydrological variation. Slightly wetter inside plots showed higher expression of denitrification genes (norB, nosZ) and lower (nirS+nirK)/nosZ ratios, indicating greater potential for complete denitrification to N2 instead of N2O. Methane dynamics were mainly associated with vegetation: plots associated with Carex rostrata exhibited lower pmoA/mcrA ratios and elevated CH fluxes. Snow manipulations had subtle effects: reduced snow depth decreased the expression of taxa dependent on microbial interactions, while the effect to the investigated metabolic marker genes was small. Overall hydrology, leading to variations in redox conditions and nutrient availability, together with vegetation appeared as the primary drivers on microbial greenhouse gas processes in this peatland.