Soil fungal community structure and function response to rhizoma perennial peanut cultivars.

BACKGROUND: Crop-associated microorganisms play a crucial role in soil nutrient cycling, and crop growth, and health. Fine-scale patterns in soil microbial community diversity and composition are commonly regulated by plant species or genotype. Despite extensive reports in different crop or its cultivar effects on the microbial community, it is uncertain how rhizoma peanut (RP, Arachis glabrata Benth.), a perennial warm-season legume forage that is well-adapted in the southern USA, affects soil microbial community across different cultivars. RESULTS: This study explored the influence of seven different RP cultivars on the taxonomic composition, diversity, and functional groups of soil fungal communities through a field trial in Marianna, Florida, Southern USA, using next-generation sequencing technique. Our results showed that the taxonomic diversity and composition of the fungal community differed significantly across RP cultivars. Alpha diversity (Shannon, Simpson, and Pielou's evenness) was significantly higher in Ecoturf but lower in UF_Peace and Florigraze compared to other cultivars (p < 0.001). Phylogenetic diversity (Faith's PD) was lowest in Latitude compared to other cultivars (p < 0.0001). The dominant phyla were Ascomycota (13.34%), Mortierellomycota (3.82%), and Basidiomycota (2.99%), which were significantly greater in Florigraze, UF_Peace, and Ecoturf, respectively. The relative abundance of Neocosmospora was markedly high (21.45%) in UF_Tito and showed large variations across cultivars. The relative abundance of the dominant genera was significantly greater in Arbrook than in other cultivars. There were also significant differences in the co-occurrence network, showing different keystone taxa and more positive correlations than the negative correlations across cultivars. FUNGuild analysis showed that the relative abundance of functional guilds including pathogenic, saprotrophic, endophytic, mycorrhizal and parasitic fungi significantly differed among cultivars. Ecoturf had the greatest relative abundance of mycorrhizal fungal group (5.10 ± 0.44), whereas UF_Peace had the greatest relative abundance of endophytic (4.52 ± 0.56) and parasitic fungi (1.67 ± 0.30) compared to other cultivars. CONCLUSIONS: Our findings provide evidence of crop cultivar's effect in shaping fine-scale fungal community patterns in legume-based forage systems.

Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

Long-term tropospheric ozone pollution disrupts plant-microbe-soil interactions in the agroecosystem.

Tropospheric ozone (O3 ) threatens agroecosystems, yet its long-term effects on intricate plant-microbe-soil interactions remain overlooked. This study employed two soybean genotypes of contrasting O3 -sensitivity grown in field plots exposed elevated O3 (eO3 ) and evaluated cause-effect relationships with their associated soil microbiomes and soil quality. Results revealed long-term eO3 effects on belowground soil microbiomes and soil health surpass damage visible on plants. Elevated O3 significantly disrupted belowground bacteria-fungi interactions, reduced fungal diversity, and altered fungal community assembly by impacting soybean physiological properties. Particularly, eO3 impacts on plant performance were significantly associated with arbuscular mycorrhizal fungi, undermining their contribution to plants, whereas eO3 increased fungal saprotroph proliferation, accelerating soil organic matter decomposition and soil carbon pool depletion. Free-living diazotrophs exhibited remarkable acclimation under eO3 , improving plant performance by enhancing nitrogen fixation. However, overarching detrimental consequences of eO3 negated this benefit. Overall, this study demonstrated long-term eO3 profoundly governed negative impacts on plant-soil-microbiota interactions, pointing to a potential crisis for agroecosystems. These findings highlight urgent needs to develop adaptive strategies to navigate future eO3 scenarios.

Ectomycorrhizal fungi enhance pine growth by stimulating iron-dependent mechanisms with trade-offs in symbiotic performance.

Iron (Fe) is crucial for metabolic functions of living organisms. Plants access occluded Fe through interactions with rhizosphere microorganisms and symbionts. Yet, the interplay between Fe addition and plant-mycorrhizal interactions, especially the molecular mechanisms underlying mycorrhiza-assisted Fe processing in plants, remains largely unexplored. We conducted mesocosms in Pinus plants inoculated with different ectomycorrhizal fungi (EMF) Suillus species under conditions with and without Fe coatings. Meta-transcriptomic, biogeochemical, and X-ray fluorescence imaging analyses were applied to investigate early-stage mycorrhizal roots. While Fe addition promoted Pinus growth, it concurrently reduced mycorrhiza formation rate, symbiosis-related metabolites in plant roots, and aboveground plant carbon and macronutrient content. This suggested potential trade-offs between Fe-enhanced plant growth and symbiotic performance. However, the extent of this trade-off may depend on interactions between host plants and EMF species. Interestingly, dual EMF species were more effective at facilitating plant Fe uptake by inducing diverse Fe-related functions than single-EMF species. This subsequently triggered various Fe-dependent physiological and biochemical processes in Pinus roots, significantly contributing to Pinus growth. However, this resulted in a greater carbon allocation to roots, relatively reducing the aboveground plant carbon content. Our study offers critical insights into how EMF communities rebalance benefits of Fe-induced effects on symbiotic partners.

Soil Bacterial Communities Across Seven Rhizoma Peanut Cultivars (Arachis glabrata Benth.) Respond to Seasonal Variation.

Soil microorganisms play key roles in soil nutrient transformations and have a notable effect on plant growth and health. Different plant genotypes can shape soil microbial patterns via the secretion of root exudates and volatiles, but it is uncertain how a difference in soil microorganisms induced by crop cultivars will respond to short-term seasonal variations. A field experiment was conducted to assess the changes in soil bacterial communities of seven rhizoma peanut (Arachis glabrata Benth, RP) cultivars across two growing seasons, April (Spring season) and October (Fall season). Soils' bacterial communities were targeted using 16S rRNA gene amplicon sequencing. Bacterial community diversity and taxonomic composition among rhizoma peanut cultivars were significantly affected by seasons, cultivars, and their interactions (p < 0.05). Alpha diversity, as estimated by the OTU richness and Simpson index, was around onefold decrease in October than in April across most of the RP cultivars, while the soils from Arblick and Latitude had around one time higher alpha diversity in both seasons compared with other cultivars. Beta diversity differed significantly in April (R = 0.073, p < 0.01) and October (R = 0.084, p < 0.01) across seven cultivars. Bacterial dominant taxa (at phylum and genus level) were strongly affected by seasons and varied towards more dominant groups that have functional potentials involved in nutrient cycling from April to October. A large shift in water availability induced by season variations in addition to host cultivar's effects can explain the observed patterns in diversity, composition, and co-occurrence of bacterial taxa. Overall, our results demonstrate an overriding effect of short-term seasonal variations on soil bacterial communities associated with different crop cultivars. The findings suggest that season-induced shifts in environmental conditions could exert stronger impacts on soil microorganisms than the finer-scale rhizosphere effect from crop cultivars, and consequently influence largely microbe-mediated soil processes and crop health in agricultural ecosystems.

Leaf Mycobiome and Mycotoxin Profile of Warm-Season Grasses Structured by Plant Species, Geography, and Apparent Black-Stroma Fungal Structure.

Grasses harbor diverse fungi, including some that produce mycotoxins or other secondary metabolites. Recently, Florida cattle farmers reported cattle illness, while the cattle were grazing on warm-season grass pastures, that was not attributable to common causes, such as nutritional imbalances or nitrate toxicity. To understand correlations between grass mycobiome and mycotoxin production, we investigated the mycobiomes associated with five prominent, perennial forage and weed grasses [Paspalum notatum Flügge, Cynodon dactylon (L.) Pers., Paspalum nicorae Parodi, Sporobolus indicus (L.) R. Br., and Andropogon virginicus (L.)] collected from six Florida pastures actively grazed by livestock. Black fungal stromata of Myriogenospora and Balansia were observed on P. notatum and S. indicus leaves and were investigated. High-throughput amplicon sequencing was applied to delineate leaf mycobiomes. Mycotoxins from P. notatum leaves were inspected using liquid chromatography-mass spectrometry (LC-MS/MS). Grass species, cultivars, and geographic localities interactively affected fungal community assemblies of asymptomatic leaves. Among the grass species, the greatest fungal richness was detected in the weed S. indicus. The black fungal structures of P. notatum leaves were dominated by the genus Myriogenospora, while those of S. indicus were codominated by the genus Balansia and a hypermycoparasitic fungus of the genus Clonostachys. When comparing mycotoxins detected in P. notatum leaves with and without M. atramentosa, emodin, an anthraquinone, was the only compound which was significantly different (P < 0.05). Understanding the leaf mycobiome and the mycotoxins it may produce in warm-season grasses has important implications for how these associations lead to secondary metabolite production and their subsequent impact on animal health. IMPORTANCE The leaf mycobiome of forage grasses can have a major impact on their mycotoxin contents of forage and subsequently affect livestock health. Despite the importance of the cattle industry in warm-climate regions, such as Florida, studies have been primarily limited to temperate forage systems. Our study provides a holistic view of leaf fungi considering epibiotic, endophytic, and hypermycoparasitic associations with five perennial, warm-season forage and weed grasses. We highlight that plant identity and geographic location interactively affect leaf fungal community composition. Yeasts appeared to be an overlooked fungal group in healthy forage mycobiomes. Furthermore, we detected high emodin quantities in the leaves of a widely planted forage species (P. notatum) whenever epibiotic fungi occurred. Our study demonstrated the importance of identifying fungal communities, ecological roles, and secondary metabolites in perennial, warm-season grasses and their potential for interfering with livestock health.

Mechanisms of stress tolerance and their effects on the ecology and evolution of mycorrhizal fungi.

Stress is ubiquitous and disrupts homeostasis, leading to damage, decreased fitness, and even death. Like other organisms, mycorrhizal fungi evolved mechanisms for stress tolerance that allow them to persist or even thrive under environmental stress. Such mechanisms can also protect their obligate plant partners, contributing to their health and survival under hostile conditions. Here we review the effects of stress and mechanisms of stress response in mycorrhizal fungi. We cover molecular and cellular aspects of stress and how stress impacts individual fitness, physiology, growth, reproduction, and interactions with plant partners, along with how some fungi evolved to tolerate hostile environmental conditions. We also address how stress and stress tolerance can lead to adaptation and have cascading effects on population- and community-level diversity. We argue that mycorrhizal fungal stress tolerance can strongly shape not only fungal and plant physiology, but also their ecology and evolution. We conclude by pointing out knowledge gaps and important future research directions required for both fully understanding stress tolerance in the mycorrhizal context and addressing ongoing environmental change.

Comparative transcriptomics of fungal endophytes in co-culture with their moss host Dicranum scoparium reveals fungal trophic lability and moss unchanged to slightly increased growth rates.

Mosses harbor fungi whose interactions within their hosts remain largely unexplored. Trophic ranges of fungal endophytes from the moss Dicranum scoparium were hypothesized to encompass saprotrophism. This moss is an ideal host to study fungal trophic lability because of its natural senescence gradient, and because it can be grown axenically. Dicranum scoparium was co-cultured with each of eight endophytic fungi isolated from naturally occurring D. scoparium. Moss growth rates, and gene expression levels (RNA sequencing) of fungi and D. scoparium, were compared between axenic and co-culture treatments. Functional lability of two fungal endophytes was tested by comparing their RNA expression levels when colonizing living vs dead gametophytes. Growth rates of D. scoparium were unchanged, or increased, when in co-culture. One fungal isolate (Hyaloscyphaceae sp.) that promoted moss growth was associated with differential expression of auxin-related genes. When grown with living vs dead gametophytes, Coniochaeta sp. switched from having upregulated carbohydrate transporter activity to upregulated oxidation-based degradation, suggesting an endophytism to saprotrophism transition. However, no such transition was detected for Hyaloscyphaceae sp. Individually, fungal endophytes did not negatively impact growth rates of D. scoparium. Our results support the long-standing hypothesis that some fungal endophytes can switch to saprotrophism.

Effects of Grass-Based Crop Rotation, Nematicide, and Irrigation on the Nematode Community in Cotton.

Plant-parasitic and free-living nematodes - bacterivores, fungivores, omnivores, predators - comprise the nematode community. Nematicide application and crop rotation are important tools to manage plant-parasitic nematodes, but effects on free-living nematodes and nematode ecological indices need further study. The nematicide fluopyram was recently introduced in cotton (Gossypium hirsutum) production and its effects on the nematode community need assessment. This research was conducted in 2017 and 2018 at a long-term field site in Quincy, FL where perennial grass/sod-based (bahiagrass, Paspalum notatum) and conventional cotton rotations were established in 2000. The objective of this research was to evaluate the effects of fluopyram nematicide, crop rotation phase, and irrigation on free-living nematodes and nematode ecological indices based on three soil sampling dates each season. We did not observe consistent effects of crop rotation phase on free-living nematodes or nematode ecological indices. Only omnivores were consistently negatively impacted by fluopyram. Nematode ecological indices reflected this negative effect by exhibiting a degraded/ stressed environmental condition relative to untreated plots. Free-living nematodes were not negatively impacted by nematicide when sod-based rotation was used.

Comparative genomics reveals dynamic genome evolution in host specialist ectomycorrhizal fungi.

While there has been significant progress characterizing the 'symbiotic toolkit' of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs) and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Correction: Metatranscriptomic Study of Common and Host-Specific Patterns of Gene Expression between Pines and Their Symbiotic Ectomycorrhizal Fungi in the Genus Suillus.

[This corrects the article DOI: 10.1371/journal.pgen.1006348.].

Reappraisal on pharmacological and mechanical treatments of heart failure.

Heart failure (HF) is a highly frequent disorder with considerable morbidity, hospitalization, and mortality; thus, it invariably places pressure on clinical and public health systems in the modern world. There have been notable advances in the definition, diagnosis, and treatment of HF, and newly developed agents and devices have been widely adopted in clinical practice. Here, this review first summarizes the current emerging therapeutic agents, including pharmacotherapy, device-based therapy, and the treatment of some common comorbidities, to improve the prognosis of HF patients. Then, we discuss and point out the commonalities and areas for improvement in current clinical studies of HF. Finally, we highlight the gaps in HF research. We are looking forward to a bright future with reduced morbidity and mortality from HF.

Correction: Metatranscriptomic Study of Common and Host-Specific Patterns of Gene Expression between Pines and Their Symbiotic Ectomycorrhizal Fungi in the Genus Suillus.

[This corrects the article DOI: 10.1371/journal.pgen.1006348.].

Nematicide influence on cotton yield and plant-parasitic nematodes in conventional and sod-based crop rotation.

Plant-parasitic nematodes (Rotylenchulus reniformis (reniform, RN), Helicotylenchus dihystera (spiral), and Mesocriconema ornatum (ring)) and yield were investigated in cotton phases of conventional (peanut-cotton-cotton) and sod-based (bahiagrass-bahiagrass-peanut-cotton) rotations with or without irrigation and fluopyram nematicide at a long-term research site, established in 2000, in Quincy, Florida, USA. Objectives were to determine impacts of nematicide application on cotton yield and evaluate effects of nematicide on plant-parasitic nematodes in these rotations in 2017 and 2018. Reniform nematode population densities were greater in conventional cotton than sod-based cotton. Ring and spiral nematode population densities were greater in sod-based cotton than conventional cotton. Plots receiving nematicide had increased RN population densities in preplant 2018 soil samples and spiral nematode population densities in preplant 2017, harvest 2017, preplant 2018, and harvest 2018 soil samples compared to untreated plots. Cotton seed yield in conventional rotation was increased by 18% following nematicide application in 2017 but decreased by 10% in sod-based rotation in 2018, relative to the untreated control. Sod-based rotation had greater cotton yield than conventional rotation in 2017 and 2018. Nematicide application did not improve cotton yield in sod-based rotation and was inconsistent in conventional rotation.Plant-parasitic nematodes (Rotylenchulus reniformis (reniform, RN), Helicotylenchus dihystera (spiral), and Mesocriconema ornatum (ring)) and yield were investigated in cotton phases of conventional (peanut­cotton­cotton) and sod-based (bahiagrass­bahiagrass­peanut­cotton) rotations with or without irrigation and fluopyram nematicide at a long-term research site, established in 2000, in Quincy, Florida, USA. Objectives were to determine impacts of nematicide application on cotton yield and evaluate effects of nematicide on plant-parasitic nematodes in these rotations in 2017 and 2018. Reniform nematode population densities were greater in conventional cotton than sod-based cotton. Ring and spiral nematode population densities were greater in sod-based cotton than conventional cotton. Plots receiving nematicide had increased RN population densities in preplant 2018 soil samples and spiral nematode population densities in preplant 2017, harvest 2017, preplant 2018, and harvest 2018 soil samples compared to untreated plots. Cotton seed yield in conventional rotation was increased by 18% following nematicide application in 2017 but decreased by 10% in sod-based rotation in 2018, relative to the untreated control. Sod-based rotation had greater cotton yield than conventional rotation in 2017 and 2018. Nematicide application did not improve cotton yield in sod-based rotation and was inconsistent in conventional rotation.

Fungal Endophytes of Populus trichocarpa Alter Host Phenotype, Gene Expression, and Rhizobiome Composition.

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.

Differential gene expression associated with fungal trophic shifts along the senescence gradient of the moss Dicranum scoparium.

Bryophytes harbour microbiomes, including diverse communities of fungi. The molecular mechanisms by which perennial mosses interact with these fungal partners along their senescence gradients are unknown, yet this is an ideal system to study variation in gene expression associated with trophic state transitions. We investigated differentially expressed genes of fungal communities and their host Dicranum scoparium across its naturally occurring senescence gradient using a metatranscriptomic approach. Higher activity of fungal nutrient-related (carbon, nitrogen, phosphorus and sulfur) transporters and Carbohydrate-Active enZyme (CAZy) genes was detected toward the bottom, partially decomposed, layer of the moss. The most prominent variation in the expression levels of fungal nutrient transporters was from inorganic nitrogen-related transporters, whereas the breakdown of organonitrogens was detected as the most enriched gene ontology term for the host D. scoparium, for those transcripts having higher expression in the partially decomposed layer. The abundance of bacterial rRNA transcripts suggested that more living members of Cyanobacteria are associated with the photosynthetic layer of D. scoparium, while members of Rhizobiales are detected throughout the gametophytes. Plant genes for specific fungal-plant communication, including defense responses, were differentially expressed, suggesting that different genetic pathways are involved in plant-microbe crosstalk in photosynthetic tissues compared to partially decomposed tissues.

Spatiotemporal Transformation in the Alkaloid Profile of Pinus Roots in Response to Mycorrhization.

Root alkaloids remain highly unexplored in ectomycorrhizae development studies. By employing ultrahigh mass resolution mass spectrometry imaging techniques, we showed substantial relocation and transformation of piperidine alkaloids in pine root tips in response to Suillus mycorrhization. We imaged, in the time frame of ectomycorrhizae formation, a completely different alkaloid profile in Pinus strobus, where basidiospores of Suillus spraguei induce morphogenesis of symbiotic tissues, than in Pinus taeda, where such interaction fails to induce morphogenesis. On the basis of spatial colocalization studies, we proposed some alternative routes for biosynthesis of these alkaloids that supplement existing literature data.

Metatranscriptomic Study of Common and Host-Specific Patterns of Gene Expression between Pines and Their Symbiotic Ectomycorrhizal Fungi in the Genus Suillus.

Ectomycorrhizal fungi (EMF) represent one of the major guilds of symbiotic fungi associated with roots of forest trees, where they function to improve plant nutrition and fitness in exchange for plant carbon. Many groups of EMF exhibit preference or specificity for different plant host genera; a good example is the genus Suillus, which grows in association with the conifer family Pinaceae. We investigated genetics of EMF host-specificity by cross-inoculating basidiospores of five species of Suillus onto ten species of Pinus, and screened them for their ability to form ectomycorrhizae. Several Suillus spp. including S. granulatus, S. spraguei, and S. americanus readily formed ectomycorrhizae (compatible reaction) with white pine hosts (subgenus Strobus), but were incompatible with other pine hosts (subgenus Pinus). Metatranscriptomic analysis of inoculated roots reveals that plant and fungus each express unique gene sets during incompatible vs. compatible pairings. The Suillus-Pinus metatranscriptomes utilize highly conserved gene regulatory pathways, including fungal G-protein signaling, secretory pathways, leucine-rich repeat and pathogen resistance proteins that are similar to those associated with host-pathogen interactions in other plant-fungal systems. Metatranscriptomic study of the combined Suillus-Pinus transcriptome has provided new insight into mechanisms of adaptation and coevolution of forest trees with their microbial community, and revealed that genetic regulation of ectomycorrhizal symbiosis utilizes universal gene regulatory pathways used by other types of fungal-plant interactions including pathogenic fungal-host interactions.

RNA-based analyses reveal fungal communities structured by a senescence gradient in the moss Dicranum scoparium and the presence of putative multi-trophic fungi.

Diverse plant-associated fungi are thought to have symbiotrophic and saprotrophic states because they can be isolated from both dead and living plant tissues. However, such tissues often are separated in time and space, and fungal activity at various stages of plant senescence is rarely assessed directly in fungal community studies. We used fungal ribosomal RNA metatranscriptomics to detect active fungal communities across a natural senescence gradient within wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution of active fungal communities in adjacent living, senescing and dead tissues. Ascomycota were active in all tissues across the senescence gradient. By contrast, Basidiomycota were prevalent and active in senescing and dead tissues. Several fungi were detected as active in living and dead tissues, suggesting their capacity for multi-trophy. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of amplicon sequencing of cDNA and metatranscriptomics is promising for studying symbiotic systems with complex microbial diversity, allowing for the simultaneous detection of their presence and activity.