HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi.

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.

Plant-associated fungi support bacterial resilience following water limitation.

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.