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.

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. Video Abstract.

Soil microorganisms help transform plant inputs into mineral-associated soil organic carbon (SOC) – the largest and slowest-cycling pool of organic carbon on land. However, the microbial traits that influence this process are widely debated. While current theory and biogeochemical models have settled on carbon-use efficiency (CUE) and growth rate as positive predictors of mineral-associated SOC, empirical tests are sparse, with contradictory observations. Using 13C-labeling of an annual grass (Avena barbata) under two moisture regimes, we found that microbial traits associated with formation of 13C-mineral-associated SOC varied by soil habitat, as did active microbial taxa and SOC chemical composition. In the rhizosphere, bacterial-dominated communities with fast growth, high biomass, and high extracellular polymeric substance (EPS) production were positively associated with 13C-mineral-associated SOC. In contrast, the detritusphere held communities dominated by fungi and more filamentous bacteria, and with greater exoenzyme activity; there, 13C-mineral-associated SOC was associated with slower microbial growth and lower microbial biomass. CUE was a negative predictor of 13C-mineral-associated SOC in both habitats. Using 13C-quantitative stable isotope probing, we found that the majority of 13C assimilation in the rhizosphere and detritusphere at week 12 of the experiment was performed by very few bacterial and fungal taxa (3–5% of the total taxa that assimilated 13C). Several complementary chemical analyses (13C-NMR, FTICR-MS, and STXM-NEXAFS) suggested that SOC in the rhizosphere had a more oxidized chemical signature, while SOC in the detritusphere had a less oxidized, more lignin-like chemical signature. Our findings challenge current theory by demonstrating that microbial traits linked with mineral-associated SOC are not universal, but vary with soil habitat and moisture conditions, and are shaped by a small number of active taxa. Emerging SOC models that explicitly reflect these interactions may better predict SOC storage, since climate change causes shifts in soil moisture regimes and the ratio of living versus decaying roots.