Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13C-AMF hyphosphere DNA from a 13CO2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13C), even though the soils’ overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera. Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.
BackgroundLinking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive to meet this goal, Stable Isotope Probing—SIP—remains the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, active microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated DNA-SIP pipeline to support well-replicated, temporally-resolved amplicon or metagenomics experiments that enable studies of dynamic microbial communities over space and time. To test this pipeline, we assembled SIP-metagenome assembled genomes (MAGs) from the hyphosphere zone surrounding arbuscular mycorrhizal fungi (AMF), in combination with a 13CO2 plant labelling study.ResultsOur semi-automated pipeline for DNA fractionation, cleanup, and nucleic acid quantification of SIP density gradients requires six times less hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients and reduced variation compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We then tested this pipeline on samples from a highly-constrained soil microhabitat with significant ecological importance, the AMF fungal hyphosphere. Processing via our quantitative SIP pipeline confirmed the AMF Rhizophagus intraradices and its associated microbiome were highly 13C enriched, even though the soils’ overall enrichment was only 1.8 atom% 13C. We assembled 212 13C-enriched hyphosphere MAGs, and the hyphosphere taxa that assimilated the most AMF-derived 13C (range 10-33 atom%) were from the phlya Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia oxidizing archaeon genus Nitrososphaeara.ConclusionsOur semi-automated SIP approach decreases operator time and errors and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. Here, we illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of active microbes living in the AMF hyphosphere (without plant roots). Their phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.
Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H218O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H218O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal–bacterial synergies may support bacterial resilience to moisture limitation.
Soil microorganisms influence the global carbon cycle by transforming plant inputs into soil organic carbon (SOC), but the microbial traits that facilitate this process are unresolved. While current theory and biogeochemical models suggest microbial carbon-use efficiency and growth rate are positive predictors of SOC, recent observations demonstrate these relationships can be positive, negative, or neutral. To parse these contradictory effects, we used a 13C-labeling experiment to test whether different microbial traits influenced the transformation of plant C into SOC within the microbial habitats surrounding living root inputs (rhizosphere) versus decaying root litter (detritusphere), under both normal soil moisture and droughted conditions. In the rhizosphere, bacterial-dominated communities with fast growth, high carbon-use efficiency, and high production of extracellular polymeric substances formed microbial-derived SOC under normal moisture conditions. However, in the detritusphere — and the rhizosphere under drought — more fungal-dominated communities with slower growth but higher exoenzyme activity formed plant-derived SOC. These findings emphasize that microbial traits linked with SOC accrual are not universal, but contingent on how microorganisms allocate carbon under different resource conditions and environmental stressors.
Drought disrupts soil microbial activity and many biogeochemical processes. Although mycorrhizal fungi are known to impact plant functions and nutrient cycling during drought, their effects on drought-exposed soil microbial communities are not well resolved. We used H218O quantitative stable isotope probing (qSIP) to investigate microbial response to water limitation in the hyphospheres of two distinct mycorrhizal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In the absence of mycorrhizal inocula, water limitation resulted in significantly lower bacterial growth rates and lower diversity in the actively growing bacterial community. Water limitation also decoupled growth from CO2 efflux, resulting in a threefold lower growth efficiency. In contrast, both mycorrhizal lineages appeared to protect bacterial communities exposed to water limitation: bacterial growth rates, growth efficiency, and the diversity of the active bacterial community were similar in water-replete and water-limited soils inoculated with either fungus. Several of the bacterial taxa that responded positively to mycorrhizal inocula in water-limited soil belong to lineages that are considered drought-susceptible. Together, these results indicate that mycorrhizal-bacterial interactions can stabilize bacterial communities in water-limited environments. As drought frequency and severity increase, these synergistic relationships may support ecosystem resilience to moisture stress.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.