Complex and stable soil microbial networks are essential for productivity in plantation forestry, but their response to disturbances from harvesting and replanting is not well understood. This study assessed the impact of treatments designed to mitigate these disturbances on microbial biodiversity and networks in Eucalyptus plantation soils at three South African sites. We used high-throughput sequencing to catalogue fungal and bacterial biodiversity from 108 soil samples across three treatments: (i) retention of harvest residues (retained), (ii) removal of residues (removed), and (iii) removal of residues with added fertilizer (fertilized). Bioinformatic and statistical analyses of the sequence data revealed treatment-specific variations in microbial OTU richness, network structure and taxon associations at order-level. Microbiome richness was highest in 'retained' plots, and treatment-specific microbial diversity was evident in both fungal and bacterial communities. Network comparisons revealed that treatments to mitigate disturbances caused by harvesting significantly affected fungal networks but not bacterial networks. Fungal networks in 'retained' plots exhibited the highest complexity and stability compared to plots where the entire crop was removed. However, bacterial networks did not show significant differences in network structure among treatments. The associations between fungal and bacterial nodes were consistent in the three treatments, as indicated by similar Jaccard indices. However, distinct fungal hub nodes were found when comparing the 'retained' and 'removed' treatments. These findings highlight that retaining harvest residues enhances microbial richness and stabilizes fungal networks, making it a better strategy for managing soil disturbances than residue removal or fertilization.

AimStudies assessing large-scale patterns of microbial diversity have predominantly focused on free-living microorganisms, often failing to link observed patterns to established theories regarding the maintenance of global diversity patterns. We aimed to determine whether foliar fungi on two closely related grass hosts-Heteropogon contortus and Themeda triandra-display a commonly observed latitudinal gradient in species richness and determine whether host identity, energy (temperature and precipitation), climate seasonality, fire frequency and grass evolutionary history drive the observed patterns in species richness and composition.LocationPaleotropical.Time PeriodContemporary.Major Taxa StudiedFoliar fungi.MethodsFoliar fungal diversity was quantified from 201 leaf samples of T. triandra and H. contortus collected across the distributional range of these species. Mixed effects models were used to quantify patterns of diversity and their correlates among and within continents. Ordinations were used to assess drivers of composition.ResultsFoliar fungi displayed consistent latitudinal diversity gradients in richness. Energy was a strong driver of richness at inter-continental and continental scales, while other factors had inconsistent impacts on richness among scales, hosts and guilds. Globally, richness was higher in regions of higher growing season temperatures and where hosts were present for longer periods. Composition was primarily structured by geographic region at the global scale, indicating that distance was a dominant driver of community composition. Within Australia, temperature and rainfall seasonality and the amount of growing season rainfall, were the dominant drivers of both richness and composition.Main ConclusionsWe find some support for the idea that foliar fungal species diversity is governed by the same factors as many macro-organisms (energy availability and evolutionary history) at inter-continental scales, but also that fungal diversity and composition in the highly seasonal continent of Australia were driven by factors that shape tropical grassy ecosystems, namely climate seasonality and fire.

RNA editing is a crucial post-transcriptional modification in endosymbiotic plant organelles, predominantly involving C-to-U conversions. Pentatricopeptide repeat (PPR) proteins play a key role in this process. To establish a system for gene expression manipulation in genetically inaccessible mitochondria, we engineered a synthetic PPR protein, dPPR-nad7-DYW, to induce de novo C-to-U editing in the NADH dehydrogenase subunit 7 (nad7) mRNA of Arabidopsis thaliana, thereby creating a premature stop codon. This designer protein, composed of 13 P-type PPR domains, was fused with the DYW-type cytidine deaminase domain from Physcomitrium patens PpPPR_56 and programmed to bind a specific nad7 mRNA segment. In vitro binding assays confirmed the specificity of dPPR-nad7-DYW for its target sequence. When expressed in Arabidopsis plants, dPPR-nad7-DYW achieved up to 85% editing efficiency at the target site, successfully introducing a premature stop codon in nad7 mRNA. This resulted in reduced polysome loading of nad7 transcripts and a phenotype characteristic of mitochondrial complex I dysfunction. RNA-sequencing revealed potential off-target editing events, albeit at lower frequencies. Our study demonstrates the successful application of an editing factor with a synthetic P-type PPR tract targeting a de novo editing site in plant mitochondria, achieving high editing efficiency. This approach opens new avenues for manipulating organellar gene expression and studying mitochondrial gene function in plants and other eukaryotes.

Xylella fastidiosa (Xf) is a Gram-negative bacterial plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its virulence is the production of extracellular vesicles (EVs). In this study, we discovered that DNA-binding proteins and nonribosomal RNA-binding proteins are abundant in the corona of Xf-EVs. DNA-seq revealed enrichment of three genomic islands (GIs) in EVs, which carry molecular signatures indicative of horizontal gene transfer (HGT). The most abundant GI encodes five homologous small RNAs designated sXFs. RNA sequencing revealed a distinct pattern of noncoding RNAs enriched in EVs, including four island-encoded sXFs. One of the sXF's stem-loops contains motifs for binding the RNA chaperone Hfq, which is also abundant in EVs. Predicted target analysis suggests that sXFs play a role in regulation of natural competence in bacteria. Additionally, sXF plant target prediction identifies a coiled-coil nucleotide-binding domain leucine-rich repeat receptor (CNL) immune gene that is downregulated following Xf infection and Xf-EV treatment. We propose a model where Xf releases nucleic acid carrying EVs with two functions: one to deliver RNA-related cargo that regulates gene expression in both bacterial and plant cells, and another to deliver DNA-related cargo for the genetic transfer of genomic islands. We highlight island-encoded sXFs as potential virulence factors and vesiduction as a mechanism of HGT of sXFs in Xf. Taken together, our data on Xf-EV cargoes provide a molecular framework for understanding the virulence of Xf.

Nodules harbour microbial communities composed of rhizobia and other lower-abundance bacteria. These non-rhizobial bacteria can promote plant growth. However, their genomic diversity and how this relates to their plant growth-promoting traits remain poorly investigated. Here, we isolated 14 Pseudomonas strains from the nodules of Lotus plants, sequenced their genomes, analysed their genomic and phylogenetic diversity, and assessed their ability to promote plant growth. We identified five distinct species, including a novel species named Pseudomonas monachiensis sp. nov., with strain PLb12AT, as the type strain. Genome analysis of these nodule-isolated Pseudomonas revealed an abundance of genes associated to plant growth-promoting traits, especially auxin-related genes, compared to closely related type strains. In accordance, most nodule-isolated Pseudomonas strains enhanced shoot growth of Lotus burttii, while only some promoted root growth or early onset of root hair proliferation. However, none of the strains significantly affected the ability to form nodules. Overall, our findings highlight the genotypic diversity and the plant growth-promoting potential of nodule-isolated Pseudomonas and underscore their possible applications in mixed inocula with rhizobia.

Fungi comprise relevant human pathogens, causing over a billion infections each year. Plastic pollution alters niches of fungi by providing trillions of artificial microhabitats, mostly in the form of microplastics, where pathogens might accumulate, thrive, and evolve. However, interactions between fungi and microplastics in nature are largely unexplored. To address this knowledge gap, we investigated the assembly, architecture, and ecology of mycobiomes in soil (micro)plastispheres near human dwellings in a model- and network-based metagenome study combined with a global-scale trait data annotation. Our results reveal a strong selection of important human pathogens, in an idiosyncratic, otherwise predominantly neutrally assembled plastisphere, which is strongly linked to generic fungal virulence traits. These findings substantiate our niche expansion postulate, demonstrate the emergence of plastiphily among fungal pathogens and imply the existence of a plastisphere virulence school, underpinning the need to declare microplastics as a factor of global health.

Coriariaceae are a small plant family of 14-17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago.

With the advances in high-throughput sequencing and bioinformatic pipelines, mitochondrial genomes have become increasingly popular for phylogenetic analyses across different clades of invertebrates. Despite the vast rise in available mitogenomic datasets of molluscs, one class of aplacophoran molluscs – Solenogastres (or Neomeniomorpha) – is still neglected. Here, we present six new mitochondrial genomes from five families of Solenogastres (Amphimeniidae, Gymnomeniidae, Proneomeniidae, Pruvotinidae, Simrothiellidae), including the first complete mitogenomes, thereby now representing three of the four traditional orders. Solenogaster mitogenomes are variable in size (ranging from approximately 15,000 bp to over 17,000 bp). The gene order of the 13 protein coding genes and two rRNA genes is conserved in three blocks, but considerable variation occurs in the order of the 22 tRNA genes. Based on phylogenetic analyses and reconstruction of ancestral mitochondrial genomes of Aculifera, the position of (1) trnD gene between atp8 and atp6, (2) trnT and P genes between atp6 and nad5, and (3) trnL1 gene between G and E, resulting in a ‘MCYWQGL1E’-block of tRNA genes, are all three considered synapomorphies for Solenogastres. The tRNA gene block ‘KARNI’ present in Polyplacophora and several conchiferan taxa is dissolved in Solenogastres. Our study shows that mitogenomes are suitable to resolve the phylogenetic relationships among Aculifera and within Solenogastres, thus presenting a cost and time efficient compromise to approach evolutionary history in these clades.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup- Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a, and 3b according to S & oslash;ndergaard, Pedersen, and Greening (HydDB: a web tool for hydrogenase classification and analysis. Sci Rep 2016;6:34212. https://doi.org/10.1038/srep34212.); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h, and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1 h and group 3b are not. Different types of [NiFe] hydrogenases in the genus Frankia and their roles in nitrogen fixation deduced from losses in lineages of genome erosion.

Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development. GENOMES UNCOUPLED1 (GUN1) plays a central role in retrograde signaling during early plant development. The putative function of GUN1 has been extensively studied, but its molecular function remains controversial. Here, we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling-relevant conditions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling. Our study of the plastid (post)transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome. By combining this result with a pentatricopeptide repeat code-based prediction and experimental validation by RNA immunoprecipitation experiments, we identified several putative targets of GUN1, including tRNAs and RNAs derived from ycf1.2, rpoC1, and rpoC2 and the ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD gene cluster. The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants account for the cotyledon phenotype. Our study provides evidence for RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies. We anticipate that our findings will serve as a basis for subsequent studies on mechanisms of plastid gene expression and will help to elucidate the function of GUN1 in retrograde signaling.

ABSTRACT Background Behavioral plasticity in the nocturnal ant Camponotus floridanus is associated with changes in daily rhythms of core clock and clock-controlled genes in the brain. Plasticity in clock-controlled output, although adaptive, has been hypothesized to be a target for parasites that change host behavior in a timely manner to complete their life cycle. This study aims to explore this hypothesis by characterizing how the transcriptomic rhythms of the ant host change upon infection by a behavior manipulating parasite. We compared and contrasted the daily gene expression profile of uninfected C. floridanus ant heads to ants infected by a manipulating fungal parasite Ophiocordyceps camponoti-floridani and a non-manipulating fungus Beauveria bassiana , to test if changes to host clock and clock-controlled gene expression are specific to behavioral modifying diseases, or if such changes are a general hallmark of infectious diseases. Results The repertoire of genes oscillating every 24h in the ant heads showed almost three-fold reduction during O. camponoti-floridani infections, as compared to uninfected controls. Control-like nocturnal activity of 24h-rhythmic genes was maintained during O. camponoti-floridani infections, but not in B. bassiana infected ant heads. Half of all genes that showed 24h rhythms in the heads and brains of uninfected ants displayed highly synchronized changes in their rhythmic expression during both diseases, but in a species-specific manner. Network analyses revealed that both fungal parasites affected the same links between behavioral plasticity and clock output, albeit in a different manner. Conclusion Changes to clock-controlled transcriptomic rhythms of hosts might be a general hallmark of infectious diseases. However, the infection-associated changes to clock-controlled rhythms of the host are species-specific, and likely depends on the life history strategies used by the parasite.

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) and whole genome sequencing of specific ticks (n = 8), we were able to determine that German samples, morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification, and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. Our results suggest that I. inopinatus is geographically isolated as a species (northern Africa and potentially southern Spain and Portugal) and brings into question whether I. inopinatus exists in central Europe. Our results highlight the probable existence of I. inopinatus and the power of utilizing genomic data in answering questions regarding tick taxonomy.

Ixodes persulcatus, a hard-bodied tick species primarily found in Asia and Eastern Europe, is a vector of pathogens to human and livestock hosts. Little research has been done on the microbiome of this species, especially using individual non-pooled samples and comparing different geographical locations. Here, we use 16S rRNA amplicon sequencing to determine the individual microbial composition of 85 Borrelia-positive I. persulcatus from the Japanese islands of Hokkaido and Honshu. The resulting data (164 unique OTUs) were further analyzed to compare the makeup and diversity of the microbiome by sex and location, as well as to determine the presence of human pathogens. We found that, while location had little influence, the diversity of I. persulcatus microbiome was predominantly dependent on sex. Males were seen to have higher microbiome diversity than females, likely due to the high presence of endosymbiotic Candidatus Lariskella arthropodarum within the female microbial communities. Furthermore, high read counts for five genera containing potentially human pathogenic species were detected among both male and female microbiomes: Ehrlichia, Borrelia, Rickettsia, Candidatus Neoehrlichia and Burkholderia and co-infections between different pathogens were frequent. We conclude that the microbiome of I. persulcatus depends mainly on sex and not geographical location and that the major difference between sexes is due to the high abundance of Ca. L. arthropodarum in females. We also stress the importance of this tick species as a vector of potential human pathogens frequently found in co-infections.

AbstractTicks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species.Ixodes inopinatusis considered a sister species and highly similar toIxodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent ofI. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified asI. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) (n=37) and whole genome sequencing of specific ticks (n=8), we were able to determine that German samples morphologically identified asI. inopinatus, genetically representI. ricinusregardless of previous morphological identification and most likely are notI. ricinus/I. inopinatushybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish betweenI. ricinusandI. inopinatus. As most examples ofI. inopinatusin Germany were based on morphology and mitochondrial sequences, the results of the current study brings into question whetherI. inopinatuswas properly identified in previous research and if this species exists in Central Europe. Our results highlight the power of utilizing genomic data in answering questions regarding tick taxonomy even when closely related species are considered.HighlightsGermanIxodes inopinatussamples representI. ricinusbased on genomic dataThe mitochondrial genome is not sufficient for delineation ofI. inopinatusandI. ricinusGerman samples most likely do not representI. ricinus/I. inopinatushybrids

Context Land use change can significantly affect plant-fungal interactions. Objectives We assessed how fungal endophytes within African wild olive ( Olea europaea subsp. cuspidata ) twigs are influenced by different levels of land use change and differences in surrounding vegetation types. Methods Twigs were sampled in the Western Cape Province (South Africa) and their fungal endophyte assemblages were characterised using culture-independent DNA metabarcoding. We assessed the effects of land use change (natural, semi-natural and planted (completely transformed)) and differences in surrounding vegetation types (grasses/low-growing plants versus shrubs/trees versus other olives) using fungal endophyte alpha and beta diversity measures. Co-occurrence networks were constructed to assess assemblage connectivity under different scenarios and to identify OTUs of potential ecological significance. Results OTU richness, but not abundance, was significantly influenced by both land use change and differences in the surrounding vegetation types. Planted African olives and those surrounded by heterospecific trees harboured the highest OTU richness. Only levels of land use change significantly influenced fungal endophyte assemblage composition. Specifically, fungal assemblages from natural habitats were distinct from those in planted and semi-natural habitats, which were similar to each other. Co-occurrence network analyses revealed that cohesive and species rich networks could only be maintained within the natural habitats. Conclusion These findings suggest that although the African olive is widespread, the identity and composition of their associated fungal assemblages are particularly sensitive to land use change. This study highlights the importance of conserving natural habitats, not just for the plants, but also for the maintenance of their associated fungal endophytes.

ABSTRACTIntroductionParasites can modify host behavior to ensure their own growth and transmission. Multiple species of the fungiOphiocordycepsinfect ants, but in a species-specific manner; one fungal species co-evolved to successfully modify the behavior of one ant species. However, several characteristics of the behavioral modification seem to be similar across differentOphiocordyceps-ant systems, including a preference for the time of the day for manipulating host behavior. In this study, we explored the various mechanisms via which the circadian clock ofOphiocordycepsmight be playing a role in modifying host behavior. We studiedO. camponoti-floridanithat modifies the behavior of its ant hostCamponotus floridanus. To separate the role of the clock in behavior manipulation, from its role in growth and survival, we compared the daily gene expression profile ofO. camponoti-floridanito a generalist, non-manipulating fungal parasite,Beauveria bassiana, which also successfully infects the same ant host.ResultsMajority of the 24h rhythmicO. camponoti-floridanigenes show peak expression before or at the transitions between light and dark. Rhythmic genes inO. camponoti-floridani, for whichB. bassianalacks an ortholog, were overrepresented for enterotoxin genes. Around half of all genes that show 24h rhythms in eitherO. camponoti-floridaniorB. bassianashowed a consistent difference in their temporal pattern of daily expression. At the halfway mark inO. camponoti-floridaniinfections, when diseased ants show a loss of 24h rhythms in daily foraging, several fungal clock genes, includingFrequency, showed differential expression. Network analyses revealed a single gene cluster, containingWhite Collar 1and2, that showed overrepresentation for genes oscillating every 24h in liquid culture as well as genes differentially expressed while growing inside the ant head.ConclusionThis study identifies several sets of putatively clock-controlled genes and biological processes inO. camponoti-floridanithat likely plays a role in modifying the behavior of its ant host. Differential expression ofO. camponoti-floridaniclock genes or 24h-rhythmic genes during infection is suggestive of either a loss of daily rhythm or a change in the amplitude of rhythmic gene expression. Both possibilities would suggest that a disease-associated change occurs to the functioning of theO. camponoti-floridaniclock, and its output, while the fungi grows inside the ant head.

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Patterns and drivers of succession provide insight into the mechanisms that govern community assembly, but remain poorly understood for microbial communities. We assess whether successional trends of trees are mirrored by foliar endophyte communities of three tree species across a deterministic woody successional gradient. Additionally, we test the relative contribution of abiotic predictors, biotic factors, and spatial distance between sites in predicting composition and richness of endophyte communities. Unlike the tree community, endophyte communities showed no consistent evidence of deterministic succession. Host identity was the most important factor structuring endophyte community composition; within hosts, spatial distance from the indigenous forest and between samples was important, while environmental predictors had small and inconsistent effects. Much variation in endophyte composition remained unexplained. In contrast, endophyte richness was well-explained by predictor variables. Host identity was most important in predicting endophyte richness, while the effect of other predictors on richness differed between host species. We conclude that deterministic succession in trees did not result in deterministic succession in endophyte communities; instead community assembly was most strongly influenced by host identity; while within hosts, neutral processes may be more important for endophyte assembly than deterministic factors.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

In forest ecosystems, decomposition is essential for carbon and nutrient cycling and therefore a key process for ecosystem functioning. During the decomposition process, litter chemistry, involved decomposer organisms, and enzymatic activity change interdependently. Chemical composition of the litter is the most complex and dynamic component in the decomposition process and therefore challenging to assess holistically. In this study, we aimed to characterize chemical shifts during decomposition and link them to changes in decomposer fungal activity. We characterized the chemical composition of freshly fallen autumn leaves of European beech (Fagus sylvatica) and the corresponding leaf litter after 1 year of decomposition by proton nuclear magnetic resonance spectroscopy. We further tested the applicability of spiking experiments for qualitative and quantitative characterization of leaves and litter chemistry. The composition and transcriptional activity of fungal communities was assessed by high-throughput Illumina sequencing in the same litter samples. We were able to distinguish freshly fallen leaves from 1-year-old litter based on their chemical composition. Chemical composition of leaves converged among regions with progressing decomposition. Fungal litter communities differed in composition among regions, but they were functionally redundant according to the expression of genes encoding litter degrading enzymes (CAZymes). Fungi of the saprotrophic genera Mycena and Chalara correlated with transcription of litter-degrading CAZymes in 1-year-old litter. Forestry measures influenced the diversity and transcription rate of the detected CAZymes transcripts in litter. Their expression was primarily predicted by composition of the soluble chemical fraction of the litter. Environmental NMR fingerprints thus proved valuable for inferring ecological contexts. We propose and discuss a holistic framework to link fungal activity, enzyme expression, and chemical composition.