Crosstalk: Interkingdom Interactions in the Mycorrhizal Hyphosphere and Ramifications for Soil Carbon Cycling
Erin E Nuccio1* (firstname.lastname@example.org), Jeff Kimbrel1, Megan Kan1, Edith Lai1, Eric Slessarev1, KJ Min2, Anne Kakouridis3, Jessica Wollard1, Marissa Lafler1, Peter Kim4, Trent Northen4, Karis McFarlane1, Mary Firestone3,4, Vanessa Brisson1, and Jennifer Pett-Ridge1
1Lawrence Livermore National Laboratory; 2Seoul National University, South Korea; 3University of California–Berkeley; and 4Lawrence Berkeley National Laboratory
Arbuscular mycorrhizal fungi (AMF) are ancient symbionts that form root associations with most plants. AMF play an important role in global nutrient and carbon cycles, and understanding their biology is crucial to predict how carbon is stored and released from soil. This Early Career research investigates the mechanisms that underpin synergistic interactions between AMF and microbes that drive nitrogen and carbon cycling, addressing DOE’s mission to understand and predict the roles of microbes in Earth’s nutrient cycles. By coupling isotope-enabled technologies with next-generation DNA sequencing techniques, this project investigates soil microbial interactions in situ using natural levels of soil complexity. This work will provide a greater mechanistic understanding needed to determine how mycorrhizal fungi influence organic matter decomposition and will shed light on nutrient cycling processes in terrestrial ecosystems.
Background: The arbuscular mycorrhizal association between Glomeromycota fungi and land plants is ancient and widespread; 72% of all land plants form symbiotic associations with AMF. While AMF are obligate symbionts that depend on host plants for C and cannot decompose soil organic matter (SOM), AMF can stimulate the decomposition of SOM and dead plant tissue. The team’s prior research strongly suggests that AMF partner with their microbiome in the zone surrounding hyphae (or “hyphosphere”) to encourage decomposition. The molecular mechanisms that underpin interactions between AMF and the microbial community during N uptake from SOM is a key knowledge gap. Researchers examine AMF-microbial interactions in both reduced complexity microcosms and in the field to assess the impact of AMF on terrestrial C and N cycling processes. In the field, team members assess how a deeply rooted perennial grass impacts soil C stocks and alters the zone of influence of AMF in soil depth profiles relative to shallow-rooted annuals.
Approach: AMF serve important roles in the soil microbial food web by stimulating soil organic matter decomposition and providing plant C to the soil community. To identify the genomes of actively growing bacteria and archaea in the AMF hyphosphere, researchers tracked plant-fixed 13CO2 through AMF hyphae into the 13C-hyphosphere microbiome using high-throughput stable isotope probing (HT-SIP) combined with high-resolution SIP-metagenomics (14 metagenomes per gradient). To separate the hyphosphere-C from the rhizosphere-C, the microcosms contained an airgap between plant and hyphal compartments that excluded roots but permitted fungal hyphae into living soil. SIP showed that the AMF Rhizophagus intraradices and associated metagenome assembled genomes (MAGs) were highly enriched (10-33 atom% 13C), even though bulk soil enrichment was low (1.8 atom% 13C). Of the 212 assembled 13C-hyphosphere MAGs, the taxa that assimilated the most AMF-13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia oxidizing archaeon genus Nitrososphaera. The phylogenetic composition and gene content of the highly 13C-enriched MAGs highlight the potential for cross-kingdom trophic interactions in the AMF hyphosphere, including predation, decomposition of fungal necromass or plant detritus, and archaeal ammonia oxidation (that may utilize ammonium or CO2 released from the aforementioned processes). In combination with other omics technologies, such as metatranscriptomics or proteomics, these MAGs will provide an important genomic resource for future experiments exploring interactions between AMF and their native microbiome.
To facilitate metabolomics and mechanistic studies of the hyphosphere, researchers have developed a sterile plant-mycorrhizal microcosm (called MycoChip, based off the EcoFAB platform) that researchers can use to interrogate hyphal-microbial interactions in situ. The MycoChip is intended to allow both destructive and nondestructive resampling of hyphosphere communities over time, and it is optically clear to permit microscopic investigation. This system has a raised airgap flanked by two 20 µm mesh barriers to create a hyphosphere zone isolated from the rhizosphere, which permits hyphae to enter the hyphae chamber but blocks root entry. The raised airgap contains a dam that prevents solute exchange between chambers in either vertical or horizontal positions. In the most recent design, the team created larger rectangular chambers that stand upright and accommodate more experimental soil in both chambers. Researchers are testing these chambers so they can be used in future experimental studies.
Most knowledge about physiology and ecology of AMF (and most soil organisms) has been learned from surface soils that are less than 20 cm deep. In a national field study, researchers assessed how the rhizosphere of a deep-rooted perennial bioenergy grass—switchgrass (Panicum virgatum)—impacts soil C stocks and alters the zone of influence of AMF and surface soil bacteria in depth profiles. Rhizosphere and bulk samples from paired switchgrass and shallow- rooted fields were collected from 2.5 m deep soil cores across nine field sites in the eastern United States (TX, MS, NC, NY, MI, WI, IL, SD). The team characterized the impact of switchgrass on soil microbial communities (AMF and bacteria), soil organic carbon (SOC), radiocarbon (14C), root abundance, and a range of soil physical and chemical properties. Switchgrass standing root biomass was significantly greater than annual standing root biomass. Across sites, radiocarbon and natural abundance 13C data suggests that switchgrass-derived C was present to a 1 m depth and ~95% of the roots were in the top 1 m. Differences in the SOC stock were highly variable, but the effect of switchgrass on SOC was more consistently positive in southerly sites featuring Alamo. The team used amplicon sequencing to characterize the AMF and bacterial communities throughout the rooting depth profiles using WANDA and 16S primer sets, respectively; this analysis will show if deeply rooted switchgrass extends the habitat of AMF down the soil profile, thus increasing their zone of influence and contribution to subsoil C cycling and weathering processes. The project’s C results indicate that standing root biomass may be a significant contributor to belowground C stocks in the first decades following conversion of annual cropland to perennial cover.
This research is supported by the U.S. Department of Energy Office of Science, Office of Biological and Environmental Research Genomic Science program under Early Career award SCW1711, DE-SC0016247 and DE-SC0020163 to UC Berkeley, and LLNL Laboratory Directed Research and Development grant 19-ERD-010. Work conducted at Lawrence Livermore National Laboratory was supported under the auspices of the U.S. DOE under Contract DE- AC52-07NA27344. Work at Lawrence Berkeley National Laboratory is supported by the m- CAFEs Microbial Community Analysis and Functional Evaluation in Soils (m-CAFEs@lbl.gov) Science Focus Area and was performed under the auspices of the U.S. Department of Energy Contract No. DE- AC02-05CH11231.