Rumen ciliate protozoa are key components of the ruminal microbiome and are considered important contributors to methane production due to their associations with methanogenic archaea. However, the diversity, evolutionary relationships, and metabolic potential of these protozoa-Archaea associations remain poorly understood. Here, we investigated the diversity and methanogenic potential of archaeal communities associated with rumen ciliates using a single-cell metagenomic approach. We analyzed genomic datasets from 22 ciliate morphospecies, comprising nine newly generated and 13 publicly available datasets, combining ribosomal marker screening, metagenomic taxonomic profiling, phylogenetic reconstruction, and functional annotation. We recovered 49 archaeal ribosomal lineages associated with 11 ciliate species, revealing considerable diversity across multiple methanogenic groups, particularly Methanobacteriales and Methanomassiliicoccales. Phylogenetic analyses revealed lineages closely related to previously characterized methanogens, as well as divergent lineages potentially representing previously uncharacterized archaeal diversity. Metagenomic profiling further expanded the detected archaeal diversity and revealed differences in archaeal community composition among ciliate hosts. Functional reconstruction of KEGG metabolic modules revealed pronounced heterogeneity in methanogenic potential, particularly in pathways associated with CO2-dependent and methylotrophic methanogenesis. While some ciliate-associated archaeal fractions exhibited extensive recovery of methanogenesis-related modules, others showed limited representation of these functions, indicating differences in the recovered metabolic repertoires among hosts. These results reveal substantial taxonomic and functional heterogeneity among rumen ciliate-associated archaeal communities and highlight the complexity of their potential contributions to ruminal methane metabolism.
The study aims to a comparative evaluation of lichens and their photobionts metabolic variation five lichen species are taken i.e., Dirinaria aegialita, Parmotrema reticulatum, Parmotrema tinctorum, Coccocarpia palmicola, and Cladonia fruticulosa, along with their isolated photobionts, Trebouxia sp. (PHB-2), Trebouxia sp. (PHB-3), Trebouxia sp. (PHB-6), Nostochopsis sp. (PHB-16), and Gloecapsa sp. (PHB-13), were investigated to compare their metabolite composition and UV-protective compounds using GCMS and LCMS analyses. GCMS profiling of lichen thalli revealed a predominance of phenolic compounds, benzoates, coumarins, aromatic metabolites, and lichen-specific polyketides, whereas the isolated photobionts were characterized by abundant saturated and unsaturated fatty acids, fatty acid methyl esters, terpenoids, sugar alcohols, amino acid derivatives, and heterocyclic compounds. These findings demonstrate clear metabolic differentiation between the lichen thalli and their cultured photobionts, indicating complementary biosynthetic functions within the lichen symbiosis. LCMS analysis of Cladonia fruticulosa detected several mycosporine-like amino acids, including shinorine, porphyra-334, palythine, palythene and palythine-threonine. Apparent quantitative analysis indicated variation in MAAs and scytonemin among the studied lichens. Overall, the metabolomic differences observed between lichen thalli and isolated photobionts highlight the photobionts as cultivable reservoirs of bioactive metabolites with promising applications in pharmaceutical, cosmetic, and nutraceutical industries.
Root-associated endophytic fungi are integral components of plant holobionts, where interspecific interactions shape ecological fitness and secondary metabolism. In this study, five fungal endophytes isolated from the roots of Salvia abrotanoides: Paraphoma radicina, Penicillium murcianum, Penicillago nodositata, Penicillium canescens, and Coniolariella hispanica were investigated to evaluate how fungal–fungal interactions influence metabolic expression under co-culture conditions. Dual-culture assays on solid and liquid media revealed distinct interaction phenotypes, including distance inhibition and contact inhibition, consistent with competitive interactions under the experimental conditions. LC–MS–based profiling of ethyl acetate extracts demonstrated that co-cultivation reshaped secondary metabolite production, leading to both suppression and induction of specific compounds. Signaling- and defense-related metabolites such as nicotinic acid, indole-3-acetic acid, and pyridoxine were differentially regulated during interaction. Notably, certain co-cultures triggered the production of metabolites absent in monocultures, including eleostearic acid and griseofulvin, indicating activation of previously silent or weakly expressed biosynthetic pathways. Among the isolates, P. nodositata was identified as a natural producer of cryptotanshinone, a diterpenoid previously associated primarily with Salvia species. However, its production was modulated under interaction conditions, suggesting context-dependent metabolic regulation. Collectively, these findings highlight the metabolic plasticity of root-associated endophytes and demonstrate that fungal–fungal interactions act as drivers of secondary metabolism reprogramming. The study underscores co-culture as an ecologically relevant strategy for uncovering interaction-mediated chemical diversity within plant-associated fungal communities.
Indigenous nodule-forming bacteria associated with French bean can differ substantially in functional phenotype, while plant responses to inoculation may also depend on external nitrogen (N) availability. This study evaluated functional variation among nodule-forming bacterial isolates recovered from French bean (Phaseolus vulgaris L.) and examined how selected isolates interacted with four mineral-N levels (0, 30, 60, and 100 kg N ha⁻¹) to influence plant growth, nodulation, N accumulation, physiological responses, and productivity. Fifty probable isolates were initially recovered from French bean nodules and tested for nodule-forming ability. Thirty-five indigenous isolates were authenticated by plant infection tests and subsequently screened in sand culture under net-house conditions. Ten comparatively effective isolates were selected for the mineral-N experiment and evaluated together with Rhizobium leguminosarum MTCC 10,096 and an uninoculated control in a completely randomized design with three biological replications. The isolates exhibited phenotypic, functional, and carbon-substrate-utilization diversity. In the pot experiment, isolate treatment, mineral-N availability, and their interaction influenced several plant traits. Increasing mineral N generally enhanced vegetative growth, biomass production, plant N accumulation, and grain yield while progressively suppressing nodulation. RD20-3 and RK3-1 were among the isolates associated with comparatively favorable responses across several plant traits, although their relative performance varied with trait and N level and nodulation of both declined as mineral N increased. Apparent inoculation-derived nitrogen gain (AING), calculated as the difference in total plant N uptake between inoculated and corresponding uninoculated treatments at the same N level, did not consistently correspond with nodule abundance. Multivariate analysis separated a dominant plant-performance/N-acquisition axis from a nodulation-dominated response axis. These results show that isolate functional differences interact with mineral-N availability to generate contrasting French bean growth, nodulation, N accumulation, and productivity responses. Because molecular identification of the indigenous isolates, nodule-occupancy confirmation, and direct N₂-fixation measurements were not undertaken, the results should not be interpreted as demonstrating taxonomic identity or isolate-specific symbiotic N₂-fixation efficiency. Graphical abstract Fig. Schematic overview of the isolation, authentication, screening, and evaluation of indigenous nodule-forming bacterial isolates associated with French bean (Phaseolus vulgaris L.) across a mineral-N gradient. Of 50 probable bacterial isolates, 35 were authenticated for nodule-forming ability by plant infection tests, and 10 indigenous isolates were selected for pot evaluation at 0, 30, 60, and 100 kg N ha⁻¹. Increasing mineral-N availability generally enhanced plant growth and N accumulation while progressively suppressing nodulation. RD20-3 and RK3-1 were associated with comparatively favorable responses across several plant traits. Multivariate analysis indicated partly distinct patterns of plant performance/N acquisition and nodulation responses. Indigenous nodule-forming isolates exhibited substantial functional and metabolic diversity. Isolate responses depended strongly on mineral-N availability in French bean. Increasing mineral N enhanced plant growth and N accumulation but progressively suppressed nodulation. RD20-3 and RK3-1 were associated with comparatively favorable responses across several plant traits. PCA separated plant performance/N acquisition from a distinct nodulation-dominated response axis.
Bromeliads play a key ecological role in many Brazilian ecosystems, particularly in campos rupestres (Brazilian rupestrian grasslands), but face increasing threats from mining and extractive activities in the Iron Quadrangle region of Minas Gerais, Brazil. Their water-filled leaf axils, or phytotelmata, host diverse microbial communities that may influence host fitness, although the ecological drivers shaping these communities remain poorly understood, especially in endangered campo rupestre ferruginoso (ferruginous rupestrian grasslands). In this study, we used 16S rRNA gene sequencing and functional prediction (PICRUSt) to compare the prokaryotic communities in the phytotelmata of two co-occurring and morphologically distinct species, Aechmea nudicaulis and Vriesea minarum, from a conserved campo rupestre ferruginoso (ferruginous rupestrian grasslands) site in Serra da Piedade (Minas Gerais, Brazil). We hypothesized that differences in plant morphology and microenvironmental conditions between host species would result in distinct microbial community structure and functional potential. Although both species shared a microbial core, significant differences in alpha diversity, community composition and predicted functional profiles, were observed. The most abundant bacterial phyla were Proteobacteria, Verrucomicrobia, and Planctomycetes, while Euryarchaeota dominated among Archaea. Environmental variables such as pH, conductivity and inorganic carbon were correlated with community composition. Notably, predicted KOs associated with nitrogen and carbon cycling pathways showed differential abundance between the two host species. These findings indicate that bromeliad phytotelmata function as host- and environment-filtered microhabitats, supporting distinct and diverse microbial communities. Given their roles in nutrient cycling, such interactions may influence plant fitness and ecosystem functioning. Understanding these relationships is important for guiding conservation efforts in fragile montane systems.
