Microbiome research has moved from cataloging community composition to asking what these communities do, but “function” is often used to describe fundamentally different levels of evidence. Functional claims may refer to functional capacity (what is encoded), functional realization (what molecular functions are actively engaged under specific conditions), or functional impact (the resulting consequences for hosts, microbial communities, or ecosystems). In this Perspective, we examine how functional annotations in microbiome research generate biological meaning and why current approaches support different kinds of inference. We propose a framework that distinguishes three levels of functional inference: capacity, realization, and impact. Homology-based, domain-centric, pathway-based, machine learning-driven, and multi-omics approaches each contribute differently to these levels, but none alone captures microbiome function in full. We argue that microbiome function should be interpreted as a hierarchy of inferences rather than a single property. Recognizing this distinction should improve how functional findings are interpreted, reported, and compared across microbiome studies. Accordingly, functional studies should explicitly state whether their conclusions concern capacity, realization, or impact, thereby clarifying the evidential basis of functional claims and improving their interpretation and comparison across microbiome studies.
Glycyrrhiza uralensis is an ecologically and economically important medicinal species for saline-alkali land restoration in arid northwest China. Nevertheless, excessive soil salinity, alkalinity and nutrient deficiency substantially restrict its growth and degrade medicinal quality. Microbial inoculation serves as a promising strategy to alleviate salt-alkali stress, yet the field stability and rhizosphere regulatory mechanisms of inoculants under diverse cultivation regimes remain poorly understood, limiting their field application. Building on our prior strain screening and preliminary field validation, this study utilized a composite inoculant containing Pseudomonas silesiensis, Arthrobacter sp. GCG3 and Rhizobium sp. DG1, and investigated its effects on G. uralensis growth, bioactive metabolites, rhizosphere soil properties and microbial communities under three field viable cultivation scenarios (direct seeding and seedling transplanting in moderately saline-alkali soil, seedling transplanting in severely saline-alkali soil) with respective tailored fertilization, planting densities and inoculation schedules. The results revealed that under all scenarios, the inoculation induced an increasing trend in the root dry weight and bioactive compound accumulation, with markedly higher dry root weight in seedling transplanting scenarios (101.68
Microplastics (MPs) represent persistent pollutants in marine environments and may act as substrates for complex microbial biofilms, collectively referred to as the plastisphere. However, the extent to which microplastic-associated communities differ from surrounding water microbiota and contribute to the environmental dissemination of potentially pathogenic taxa and antibiotic resistance genes (ARGs) remains incompletely understood. In this study, we investigated environmental MPs collected in the Northern Adriatic Sea together with polyethylene MPs experimentally aged for eight months under mesocosm conditions. Using 16S rRNA gene amplicon sequencing, shotgun metagenomics and droplet digital PCR, we compared MP-associated biofilms with the corresponding marine and lagoon water microbiota and assessed selected ARG profiles. Microplastic-associated biofilms displayed significantly lower alpha-diversity and distinct beta-diversity patterns compared with free-living communities, indicating that MPs harboured microbial communities differing from the surrounding water microbiota. Both environmental and experimentally aged MPs showed a higher relative presence of potentially pathogenic genera, with Vibrio representing the dominant genus within the potentially pathogenic component of the plastic microbiota. Shotgun metagenomic analysis revealed a richer and more diverse resistome on environmental MPs compared to seawater, including resistance classes exclusively detected on MPs. Digital droplet PCR confirmed higher normalized abundances of key ARGs (tetA, tetB, sul2) on MPs, together with the preferential detection of clinically relevant genes such as mcr-1 and qnr variants. By integrating environmental MPs, experimentally aged polyethylene MPs, 16S rRNA gene amplicon sequencing, shotgun metagenomics and ddPCR validation, this study provides a combined environmental and experimental assessment of MP-associated microbial communities and selected ARGs in marine and lagoon environments. Together, these findings demonstrate that marine MPs may act as persistent biologically active substrates for biofilm-forming microbial communities that differ from surrounding free-living communities and may contribute to the spread of ARGs, highlighting the need for integrated monitoring of MPs pollution and antimicrobial resistance.
Plant disease complexes are difficult to resolve because disease progression can be correlated among multiple microbial taxa within the same host environment rather than arising from a single causal agent. Cranberry fruit rot (CFR), one of the most economically important diseases in cranberry production, has been associated with up to 15 fungal species. Current disease etiology describes an initial floral infection followed by a persistent latent period until disease development in mature fruits. However, this model has not been demonstrated, and it is still unknown whether it is applicable to all fungal species. We conducted a two-year field study of the cranberry fruit endophytic mycobiome across successive phenological stages. Combining ITS2 amplicon sequencing with detection-probability modeling and quantitative PCR–based estimates of fungal biomass and species-specific detection, we evaluated mycobiome dynamics with a particular focus on disease-associated species. Host phenology explained most of the variation in fungal richness and community composition. CFR-associated fungi were infrequently detected during flowering and fruit set and occurred at very low biomass, but their prevalence and abundance increased markedly during fruit maturation. These taxa became dominant in asymptomatic ripe berries and were further enriched in rotten fruit. Targeted qPCR assays corroborated the near absence of major CFR-associated fungi at early developmental stages, inconsistent with the widely proposed model of flowering-time infection. Our data support a framework in which disease emergence is linked to a phenology-dependent reorganization of the resident mycobiome accompanied by a strong increase in fungal biomass. This shift in community structure revises the current view of CFR epidemiology and underscores the role of host developmental filtering in structuring pathogen-complex dynamics in perennial crops. In addition, this pathosystem could serve as a model system for testing community-based models of pathogenesis in perennial crops.
The leaf surface, or phyllosphere, hosts abundant and diverse bacterial communities that interact with both the host plant and herbivorous insects, yet their collective influence on plant–insect interactions remains poorly investigated. We established a functionally gnotobiotic insect–plant system combining axenically grown Arabidopsis thaliana, Pieris brassicae larvae, and defined synthetic phyllosphere communities (SynComs) of increasing richness: SynCom5, SynCom10 and SynCom20, containing 5, 10 and 20 bacterial strains, respectively. We investigated how phyllosphere bacteria influence herbivore performance, plant defence responses, and bacterial colonisation of both leaves and the insect gut. While larval weight tended to decrease with increasing bacterial community richness, only the most diverse SynCom (20 members) caused significant weight reductions without affecting survival. Plants harbouring the most diverse community showed enhanced jasmonic acid (JA) levels during feeding, whereas salicylic acid (SA) remained unchanged, suggesting the induction of herbivore-associated defence responses. Compared to plants experiencing no herbivory, feeding strongly reshaped bacterial colonisation on leaves, increasing total bacterial loads about fourfold and driving dominance of Pantoea eucalypti 299R as shown by 16S rRNA gene amplicon sequencing. Larvae acquired a distinct subset of bacteria, primarily recruited from the genera Methylobacterium, Microbacterium, Williamsia, and Curtobacterium. Phyllosphere bacteria can influence plant–insect interactions, with bacterial community richness associated with reduced P. brassicae larval performance and SynCom20 linked to stronger JA accumulation in leaves during feeding. Conversely, herbivory increased leaf bacterial loads, shifted leaf community composition, and filtered leaf-associated bacteria during passage into the larval gut. These findings establish this functionally gnotobiotic insect–plant system as a tractable model for dissecting reciprocal effects between resident phyllosphere bacteria and herbivory.
Ongoing climate change drives drought events that threaten crop yields worldwide. Traditionally, the focus has been on plant breeding to mitigate losses due to stress; however, this approach fails to leverage the resources available within the holobiont. Microbial treatments have been shown to confer drought tolerance by altering phytohormone levels, enhancing nutrient acquisition, and improving water retention in their plant hosts. However, the role of core seed-associated endophytic fungi in stress resilience remains underexplored. Using Moesziomyces antarcticus RS1, which was previously identified as a core fungus in wild and domesticated rice seed microbiomes, we revealed the functional roles of a core microbe in the host plant’s physiology. M. antarcticus RS1 displayed nutrient- and osmotic stress-responsive dimorphism, consistent with adaptive persistence in the seed endosphere. M. antarcticus RS1 inoculation on rice seed significantly improved yield traits by increasing grain quantity and biomass. Notably, M. antarcticus RS1 seed-inoculated rice performed better under PEG-induced osmotic stress, which was associated with increased osmoprotectant levels and growth benefits observed under PEG-treated and untreated conditions. Treatment of M. antarcticus RS1 was associated with increases in drought-related class 2 and class 3 Late Embryogenesis Abundant gene expression in rice, including a suite of RAB dehydrins. These findings establish M. antarcticus RS1 as a core seed endophyte that improves rice drought tolerance and productivity, positioning it as a novel bioinoculant for sustainable agriculture, particularly in water-limited environments.
Interactions between tree root exudates and rhizosphere bacteria are a key pathway influencing tree health in the forest. However, how this interaction occurs at the chemical level belowground in forest soil still remains unclear. We conducted a 2-year field experiment in a mixed Mediterranean forest with mature trees of Pinus halepensis, Quercus calliprinos and Pistacia lentiscus to examine the interplay between tree roots and bacterial community dynamics under seasonal drought. Root exudate rate and metabolite composition were analyzed simultaneously with 16S rRNA sequencing to the species level of soil and root bacterial communities across four seasons. Root exudates increased, on average, 3.3-fold in the dry vs. wet season across the three species, reaching up to 41.7 μg C day−1 cm−2. Eighty-nine metabolites were responsive to seasonal drought, consisting largely of amino acids (24), phenolics (21), carbohydrates (11) and terpenoids (9). Across all tree species, eight metabolites were drought-responsive. Metabolite composition varied with tree species and season. However, root bacterial communities changed with host tree species but not with season. Multiple bacterial taxa correlated either positively or negatively with drought-responsive metabolites; for example, 19 actinobacteria species were associated with compounds such as the terpenoid glaucocalyxin A and the carbohydrates deoxyribose and a C5 sugar alcohol, illustrating diverse bacterial responses to seasonal drought-induced exudation shifts. Our results suggest that in a Mediterranean forest experiencing recurrent seasonal drought, drought-induced shifts in root exudate composition may act as metabolic cues that are associated with changes in the rhizosphere bacterial community, while preserving its overall taxonomic stability.
Microplastics have become a global environmental concern, but the impact of microplastics on plant disease remains poorly understood. The primary objective of this study was to evaluate the effects of low-density polyethylene (LDPE) microplastics on cotton Verticillium wilt (CVW), as well as the structure and function of the soil microbial community. Metagenomic sequencing was performed to characterize the microbial community composition of the cotton rhizosphere. The experiment consisted of five treatments: a control group, and four LDPE groups with concentrations set at 0.1
Cell size regulates the evolution and selection of bacterial metabolic strategies through the "resource acquisition capacity—metabolic cost trade-off", resulting in significant differences in community and metabolic functions among bacteria with different size. Therefore, phenotypic grouping of bacterial communities based on cell size can directly establish the mechanism connection between the "phenotypic structure" of microbial communities and ecological function, which has significant theoretical significance for clarifying the role and status of bacteria of different cell sizes in the biogeochemical cycle process. The results showed that UB and LB were clearly distinguishable in the environment, and their relative proportions varied with environmental conditions. Compared with LB, UB exhibited lower diversity and narrower ecological niche breadth, the community compositions of the two groups showed significant differences, leading to distinct ecological functions. Notably, UB play a crucial role in material cycling, contributing to the maintenance of ecosystem balance and stability, and participating actively in elemental cycles. Our findings highlight the utility of cell size as an indicator of bacterial diversity and reveal a direct link between microbial morphology and ecological characteristics.
Phages shape microbial communities by regulating metabolic pathways, driving biogeochemical processes, and impacting stability and functionality of ecosystems. Phages contributions to natural ecosystems are undeniable; however, their role in built systems especially in anaerobic digesters remain poorly characterized. To discover the functional role of phage communities in anaerobic digesters, we evaluated phage-bacterial and virus-archaeal relationships in metagenomic sequences from fifteen commercial, full-scale anaerobic digesters of chicken, cattle, and pig manure, the three most commonly utilized organic waste streams globally. Here, we predict the abundance, auxiliary metabolic genes, and microbial-host interactions of phage and archaeal viruses under anaerobic fermentation processes and methanogenesis. We found phages and prokaryote abundances were coupled and both populations were driven by feedstock characteristics (20
Alpine freshwater habitats are biodiversity hotspots, home to uniquely adapted microbial communities. However, our understanding of how taxonomic composition and metabolic potential of microbial communities varies across habitat type and physiography remains limited. Using metagenomic sequencing, we examined microbial diversity and functions in sediment and water habitats across a physiographic gradient, including deep and shallow lakes, pasture ponds, ponds and peat bogs. Overall, sediments supported more diverse and even microbial communities. Conversely, waters displayed higher heterogeneity across the physiographic gradient in both taxonomic and functional composition. Notably, water communities from small water bodies, especially peat bogs and ponds, showed comparable diversity to their respective sediment communities, with higher taxonomic richness than aquatic communities from larger water bodies. Sediment microbial communities showed higher functional potential for nitrogen and methane metabolism, a pattern related to the likely anoxic conditions. By contrast, water bodies favored higher potential for carbohydrate metabolism, carbon oxidation and photosynthesis. Lastly, we recovered 496 bacterial and archaeal metagenome-assembled genomes and found that 73
Dispersants are commonly used worldwide as a primary response tool to treat oil spills at sea, yet their use is debated due to their toxicological effects and potential to affect oil biodegradation rates. We examined the effect of three globally stockpiled synthetic chemical dispersants (Superdispersant 25, Slickgone EW, Slickgone NS) on the microbial response to crude oil and its biodegradation in a subarctic marine environment in two experiments using seawater sampled from the subarctic Faroe-Shetland Channel (FSC) in 2015 and 2017 and analysed by 16 S rRNA gene amplicon sequencing. Across both years, communities were dominated by known hydrocarbon-degrading bacteria, including Alcanivorax, Alteromonas, and Pseudoalteromonas. In the 2015 experiment, Superdispersant 25 enriched for Dokdonia, Thalassospira, and Roseobacter, genera with demonstrated alkane and PAH degradation pathways. In the 2017 experiment, Slickgone EW and NS selected Marinomonas, Colwellia, Psychromonas, and Alcanivorax. Samples without dispersant also contained hydrocarbon degraders, however, the community composition was altered. In 2017, we quantified hydrocarbon degradation using GC-FID/MS for aliphatic and aromatic hydrocarbons respectively. Hydrocarbon biomarker ratios showed n-alkane depletion (Pr/C17 and Ph/C18 ratios increased 3–12 fold) in dispersant treatments. Selective weathering was evident in all treatments. 9MP/1MP ratios indicated limited or variable effects on aromatic hydrocarbon biodegradation, particularly in the presence of dispersant. We integrated aromatic hydrocarbon concentrations with microbial community data using DIABLO (Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies). Several taxa were negatively correlated to specific aromatic hydrocarbons – i.e. they increased when the hydrocarbon was reduced suggesting the hydrocarbons may have served as a carbon source or the taxa were responding to hydrocarbon depletion. Taxa included Aquibacter, Hyphomonas, Thalassospira, Alteromonas, Sphingorhabdus, and Paraperlucidibaca. Marine oil snow (MOS) formed in all oil-amended treatments and showed high microbial colonisation, whereas marine dispersant snow showed little colonisation. This study provides evidence that different dispersants affect different microbial responses to crude oil contamination, including the enrichment of key oil-degrading bacterial taxa. However, this did not correlate with enhanced aromatic hydrocarbon degradation. MOS formation with high microbial colonisation suggests natural aggregation processes may provide an effective biodegradation pathway. These findings raise questions about the functional benefit of synthetic chemical dispersants and highlight that the marine environment naturally harbours hydrocarbon-degrading microbial communities with the potential to respond to oil spills.
Pelagic Sargassum has undergone significant range expansion and dramatic blooms in the Atlantic over the past 15 years. This alga’s microbiome provides symbiotic functions that are believed to contribute to its ecological success. Recent research shows that Sargassum-associated bacteria are enriched in integrated prophages compared to the surrounding seawater and that these prophages are inducible by chemical and ultraviolet treatment. Here, we investigated a Sargassum-derived in vitro multispecies biofilm encompassing the dominant heterotrophic microbial members associated with Sargassum to probe the impacts of prophage induction on the composition of Sargassum microbiomes. Induction was quantified by coverage-based virus-to-host ratios in chemically induced treatments with Mitomycin C and non-induced controls, and the community composition and metabolic profiles were analyzed after Mitomycin C treatment. Chemical induction led to a significant increase in abundance and virus-to-host ratio of viral genomes linked to Vibrio metagenome-assembled genomes. This was accompanied by altered biofilm community composition, with a reduction in Vibrio bacterial abundance that opened niche space for other biofilm members in the genera Pseudoalteromonas, Alteromonas, and Cobetia. The induced Vibrio-associated phages encoded genes involved in quorum sensing, biofilm formation, virulence, and host metabolism. Induction led to the depletion of 17 metabolic modules, including functions related to energy metabolism and nitrogen utilization. Due to the high frequency of lysogeny in the Sargassum microbiome and the susceptibility of prophages to chemical and ultraviolet light induction, these results suggest that prophage integration and induction are mechanisms that contribute to structuring the Sargassum microbiome and its functional profiles, potentially aiding in microbiome flexibility in changing environmental contexts.
Understanding ecosystem dynamics is essential for assessing ecosystem health, yet remains challenging due to complex biotic and abiotic interactions. Microbial communities are valuable indicators of environmental change, but the high dimensionality of microbiome data requires advanced analytical methods. This study explores the use of topic modeling (TM), an unsupervised machine learning approach initially designed for text analysis, to analyze microbiome data from the dynamic Warnow Estuary on the southern Baltic Sea coast. We applied TM to estuarine microbiome data and compared its performance to traditional dimensionality reduction methods, Principal Component Analysis (PCA) and Principal Coordinate Analysis (PCoA). Quantitative results indicate that TM performs comparably to conventional approaches in preserving ecological and functional information, and in certain aspects even superior. In addition, we show qualitatively that Non-Negative Matrix Factorization (NNMF), a TM method, captures latent patterns in the data providing an interpretable perspective on the microbiome. In this exploratory framework, NNMF suggested five distinct sub-communities within the estuary that appear to follow a seasonal succession influenced by freshwater inflow. These sub-communities were associated with specific ranges of salinity and temperature and showed distinct taxonomic profiles, with shared characteristics across the estuarine system. Our findings suggest that TM is a useful tool for exploring complex environmental microbiome datasets, offering a complementary perspective that can provide additional ecological insights. TM’s ability to highlight coherent microbial community patterns indicates its promise for supporting environmental monitoring and informing targeted ecosystem management in dynamic habitats, though further studies are needed to fully assess its applicability.
The phycosphere is a nutrient-rich microenvironment surrounding phytoplankton cells and serves as a hotspot for microbial interactions by releasing phytoplankton-derived organic molecules to dynamically attract and support the colonization of heterotrophic bacteria. Although the taxonomic composition, metabolic profiles, and host-microbe interactions of phycosphere bacterial communities have been extensively characterized, the underlying mechanisms driving competition among these co-existing bacterial taxa remain poorly understood. In this study, we demonstrate that Bacillus velezensis SPE2, a low-abundance isolate from the phycosphere of dinoflagellate, exhibits a wide degree of antagonistic activity against multiple marine Flavobacteriaceae strains, a dominant taxonomic group across the phycosphere of diverse phytoplankton species. Through an integrative approach combining genetics and metabolomics, we show that the antagonistic behavior of strain SPE2 is primarily mediated by the production of bioactive secondary metabolites. Activity-guided purification further leads to the identification of two antibacterial surfactin-like lipopeptides as key exometabolites responsible for these inhibitory effects. Mechanistically, these lipopeptides exert their antibacterial activity against Flavobacteriaceae species by disrupting the integrity of bacterial cell membranes. Our findings reveal surfactin-like lipopeptides as key molecular mediators of bacterial interference competition, conferring a competitive strategy for Bacillus species to secure persistence in the phycosphere. Moreover, this work underscores phycosphere as a largely untapped ecological niche for discovering novel bioactive compounds with potential applications in pharmaceutical and biotechnological fields.
The functional responses of soil microbiomes to concurrent warming and altered precipitation in alpine deserts remain poorly understood, hindering predictions of these fragile ecosystem to climate change. Specifically, the mechanisms by which microbial communities maintain ecosystem function potential despite climate-induced biodiversity changes are unclear. A three-year field manipulation experiment in an alpine desert grassland on the Qinghai-Xizang Plateau showed that warming and watering acted as distinct ecological drivers. Warming restructured prokaryotic and fungal communities, favored stress-associated taxa, and increasing interkingdom network complexity, indicating tighter microbial associations under climate stress. Although warming reduced microbial richness and diversity, it did not diminish the overall potential for soil nutrient cycling. Instead, functional stability was associated with sustained microbial abundance, network reorganization, and selective changes in nutrient-cycling genes, particularly those involved in nitrogen and phosphorus transformation hosted by specific bacterial phyla. In contrast, watering did not significantly increase mean soil moisture, but altered soil nutrient availability, affecting key microbial groups and their functions, showing an indirect regulation pathway. Functional stability in alpine deserts under climate change was maintained not by taxonomic diversity alone, but through abundance-based compensation, community reorganization, and pathway-specific functional shifts. This study provides a mechanistic framework linking climate drivers to microbial community structure and nutrient-cycling potential, offering predictive insights into the responses of cold-arid ecosystems to future climate change.
Hardwood tree growth and development are influenced by the interplay between soil microbiota and tree physiology. Though unclear mechanistically, microbial community composition is associated with tree growth. We investigated how soil chemistry and prokaryotic communities impacted Quercus rubra (red oak) and Juglans nigra (black walnut) growth across three plantations in Indiana and Michigan. Trees exhibiting above- (high-performing or healthy) or below-average (low-performing or poor) growth rates were compared. Correlations with soil properties and microbial composition (16 S rRNA sequencing) were assessed to identify factors associated with variations in mean dominant tree height and diameter at breast height (DBH). Tree growth differed by site, species, and performance class. High-performing trees showed 25–40
Background The composition of the seed-associated bacterial microbiome can reflect host evolutionary relationships, a pattern consistent with phylosymbiosis. While machine learning offers new opportunities to predict microbial community composition, existing models often require prior microbial profiles or environmental variables, limiting their application to unsampled hosts. Here, we tested whether plant nuclear internal transcribed spacer (ITS) sequences, used as a marker of host relatedness, can predict species-level seed-associated bacterial communities using 16S rRNA data from 61 plant species.Results We introduced customized machine learning models that use sequence-based Hamming distances to capture plant host relatedness. Among the tested models, the Hamming Distance-based k-Nearest Neighbor model (HD-KNN) achieved the highest overall predictive accuracy, yielding an average Jensen-Shannon divergence (JSD) of 0.276 between observed and predicted microbiome profiles. HD-KNN performed particularly well within densely sampled host groups, including Brassicaceae and Poaceae, where closely related reference species were available. In contrast, Hamming Distance-based Gaussian Process Regression (HD-GPR) showed slightly better performance for phylogenetically isolated species, suggesting that model performance depends on host representation within the training dataset.Conclusions Our framework demonstrates that plant nuclear ITS-derived host relatedness carries a partial predictive signal for seed-associated bacterial microbiome composition. These results provide a foundation for low-input predictive modelling of seed-associated bacteria and may help prioritise microbiome predictions for unsampled plant species when closely related reference species are available. However, our conclusions are strictly limited to seed-associated bacterial communities and should not be directly generalized to fungal communities or other plant compartments, such as the rhizosphere or phyllosphere, which may be shaped by different environmental filtering mechanisms.
Tree seeds harbor diverse fungal communities, including both pathogens and mutualists, that can influence plant health. These communities comprise living, metabolically active organisms as well as dormant or dead cells. Because only active fungi interact with their hosts, distinguishing active from inactive taxa is crucial, especially for environmental and phytosanitary monitoring. Traditional culturing methods capture living fungi but account for only a small fraction of the total fungal diversity. Currently, these methods are increasingly replaced by high-throughput DNA metabarcoding, which detects a broader range of taxa. However, DNA persists after cell death and occurs in dormant cells, preventing distinction between active and inactive fungi. In contrast, RNA metabarcoding may better reflect living fungal communities than the other two methods, though its use in assessing plant-associated fungi remains underexplored. We used culturing, DNA-, and RNA-based metabarcoding to compare fungal communities associated with seeds of three key European tree species (Fagus sylvatica, Abies alba, Pinus sylvestris). Dominant fungal communities in seeds were strongly shaped by host species identity and were largely shared across DNA and RNA metabarcoding datasets, with roughly half of the most abundant genera detected by both methods. Differences between DNA- and RNA-derived communities were predominantly associated with rare taxa in the RNA dataset, although distinguishing true biological signals from noise introduced by different methodological workflows remains challenging. Several cultured genera, likely both abundant and metabolically active, were consistently detected by both approaches. These results highlight the complementary nature of the three methods for characterising seed-associated fungi. Combining culturing, DNA- and RNA-based metabarcoding may provide the most comprehensive assessment of fungal diversity, while RNA metabarcoding alone offers a promising opportunity to identify the active members of fungal communities for improved environmental and phytosanitary monitoring.
Phyllosphere microorganisms on leaf surfaces play key roles in maintaining plant productivity and health. The composition of phyllosphere microbial communities may vary during leaf aging, leading to changes in the complexity and stability of these communities. However, the ecological mechanisms underlying these varistions, particularly the relative contributions of deterministic (i.e., environmental filtering) and stochastic assembly processes (i.e., random dispersal and ecological drift) in shaping community dynamics, remain unclear. In this study, we collected 252 needle samples corresponding to three age cohorts from three representative evergreen coniferous species in natural mixed broadleaved-Korean pine forests throughout Northeast China. Needle traits were determined, and both phyllosphere epiphytic bacterial and fungal communities were assessed using amplicon sequencing. Our results showed that stochastic assembly dominated microbial communities, and the longer immigration periods contributed to increased microbial diversity and network complexity across needle age cohorts. Deterministic assembly became stronger for both bacterial and fungal communities during needle aging, which was closely related to the increased concentrations of secondary metabolites such as flavonoids, resulting in gradually elevated community similarity. Microbial network stability decreased in older needles, primarily due to the lower modularity and fewer negative interactions among species. Our study provides comprehensive empirical evidence on the succession of phyllosphere bacterial and fungal communities across needle age cohorts, emphasizing the importance of a dynamic balance between deterministic assembly and stochastic assembly in shaping microbial communities during needle aging.
