The reference genome and abiotic stress responses of the model perennial grass Brachypodium sylvaticum.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.

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.

What are mycorrhizal traits?

Traits are inherent properties of organisms, but how are they defined for organismal networks such as mycorrhizal symbioses? Mycorrhizal symbioses are complex and diverse belowground symbioses between plants and fungi that have proved challenging to fit into a unified and coherent trait framework. We propose an inclusive mycorrhizal trait framework that classifies traits as morphological, physiological, and phenological features that have functional implications for the symbiosis. We further classify mycorrhizal traits by location - plant, fungus, or the symbiosis - which highlights new questions in trait-based mycorrhizal ecology designed to charge and challenge the scientific community. This new framework is an opportunity for researchers to interrogate their data to identify novel insights and gaps in our understanding of mycorrhizal symbioses.

Life and death in the soil microbiome: how ecological processes influence biogeochemistry.

Soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter - Earth's largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

Label-Free Multiphoton Imaging of Microbes in Root, Mineral, and Soil Matrices with Time-Gated Coherent Raman and Fluorescence Lifetime Imaging.

Imaging biogeochemical interactions in complex microbial systems─such as those at the soil-root interface─is crucial to studies of climate, agriculture, and environmental health but complicated by the three-dimensional (3D) juxtaposition of materials with a wide range of optical properties. We developed a label-free multiphoton nonlinear imaging approach to provide contrast and chemical information for soil microorganisms in roots and minerals with epi-illumination by simultaneously imaging two-photon excitation fluorescence (TPEF), coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and sum-frequency mixing (SFM). We used fluorescence lifetime imaging (FLIM) and time gating to correct CARS for the autofluorescence background native to soil particles and fungal hyphae (TG-CARS) using time-correlated single-photon counting (TCSPC). We combined TPEF, TG-CARS, and FLIM to maximize image contrast for live fungi and bacteria in roots and soil matrices without fluorescence labeling. Using this instrument, we imaged symbiotic arbuscular mycorrhizal fungi (AMF) structures within unstained plant roots in 3D to 60 µm depth. High-quality imaging was possible at up to 30 µm depth in a clay particle matrix and at 15 µm in complex soil preparation. TG-CARS allowed us to identify previously unknown lipid droplets in the symbiotic fungus, Serendipita bescii. We also visualized unstained putative bacteria associated with the roots of Brachypodium distachyon in a soil microcosm. Our results show that this multimodal approach holds significant promise for rhizosphere and soil science research.

Plants and mycorrhizal symbionts acquire substantial soil nitrogen from gaseous ammonia transport.

Nitrogen (N) is an essential nutrient that limits plant growth in many ecosystems. Here we investigate an overlooked component of the terrestrial N cycle - subsurface ammonia (NH3 ) gas transport and its contribution to plant and mycorrhizal N acquisition. We used controlled mesocosms, soil incubations, stable isotopes, and imaging to investigate edaphic drivers of NH3 gas efflux, track lateral subsurface N transport originating from 15 NH3 gas or 15 N-enriched organic matter, and assess plant and mycorrhizal N assimilation from this gaseous transport pathway. NH3 is released from soil organic matter, travels belowground, and contributes to root and fungal N content. Abiotic soil properties (pH and texture) influence the quantity of NH3 available for subsurface transport. Mutualisms with arbuscular mycorrhizal (AM) fungi can substantially increase plant NH3 -N uptake. The grass Brachypodium distachyon acquired 6-9% of total plant N from organic matter-N that traveled as a gas belowground. Colonization by the AM fungus Rhizophagus irregularis was associated with a two-fold increase in total plant N acquisition from subsurface NH3 gas. NH3 gas transport and uptake pathways may be fundamentally different from those of more commonly studied soil N species and warrant further research.

Widespread heterogeneity in staple crop mineral concentration in Uganda partially driven by soil characteristics.

Calcium (Ca), iron (Fe), selenium (Se), and zinc (Zn) deficiencies are widespread in sub-Saharan Africa, with severe implications for human health. In Uganda, where the predominant diet depends heavily on plant-based staples, crop mineral concentration is an important component of dietary mineral intake. Studies assessing the risk of nutrient deficiency or the effectiveness of nutrient-focused interventions often estimate dietary mineral intake using food composition tables that are based on crops grown in developed countries. However, little is known about the actual nutritional content of crops grown in Uganda. Here, we document the Ca, Fe, Se, and Zn concentration of staple crops collected from Ugandan household farms. While median mineral concentrations were similar to those reported previously, variation in crop mineral concentration was high, particularly for Fe and Se. An ordinary least squares regression showed that some soil characteristics were correlated with crop mineral concentrations. Of these, soil pH was often positively associated with crop mineral concentration, while sand and organic carbon concentrations were negatively associated with several crop mineral concentrations. However, much of the variation in crop mineral content was not associated with the soil characteristics measured. Overall, our results suggest that extensive heterogeneity in staple crop mineral concentration in Uganda is likely due to a combination of edaphic characteristics and other variables. Because staple foods constitute a large portion of dietary mineral intake in Uganda and other developing countries, these results have implications for estimates of dietary mineral intake and the development of effective intervention strategies in such regions.

Ammonia volatilization from composting with oxidized biochar.

Animal manure, agricultural residues, and other sources of biomass can be diverted from the waste stream and composted into valuable fertilizer. However, composting often results in substantial N loss through NH3 gas volatilization. We investigated biochar's capacity to improve NH3 -N retention during composting of poultry manure and straw. After 7 wk, total N loss from composting with unoxidized biochar was twofold and sixfold higher than N loss from composting with oxidized biochar and without biochar (307, 142, and 51 mg N g-1 N in the initial compost feedstocks, respectively). When cumulative NH3 -N loss was calculated relative to CO2 -C loss to account for differences in microbial activity, NH3 -N/CO2 -C loss from compost with oxidized biochar was 55% lower than from compost with unoxidized biochar (82% lower based on mass balance). Oxidized biochar particles removed from compost after 7 wk retained 16.0 mg N g-1 biochar, compared with only 6.1 mg N g-1 retained by unoxidized biochar, suggesting that N retention by biochar particles provides a mechanism for reduced NH3 -N loss. These data show that oxidized biochar enhanced microbial activity, doubled composting rate, and reduced NH3 -N loss compared with unoxidized biochar and that biochar's physiochemical characteristics modulate its performance in compost. In particular, the presence of oxidized surface functional groups, which can be increased artificially or through environmental weathering, appear to play an important role in key compost processes. This has implications for other natural and managed systems where pyrogenic organic matter may mediate biological activity and nutrient cycles.

Variation in crop zinc concentration influences estimates of dietary Zn inadequacy.

BACKGROUND: Zinc (Zn) deficiency is one of the most common micronutrient deficiencies worldwide. Accurate estimates of Zn intake would facilitate the design and implementation of effective nutritional interventions. OBJECTIVE: We sought to improve estimates of dietary Zn intake by evaluating staple crop Zn content and dietary Zn consumption by children under the age of 5 in 9 rural districts of Uganda. METHODS: We measured the Zn content of 581 crop samples from household farms and 167 crop samples from nearby markets, and administered food frequency questionnaires to the primary caretakers of 237 children. We estimated Zn consumption using 3 sources of crop Zn content: (i) the HarvestPlus food composition table (FCT) for Uganda, (ii) measurements from household crops, and (iii) measurements from market crops. RESULTS: The Zn content of staple crops varied widely, resulting in significantly different estimates of dietary Zn intake. 41% of children appeared to be at risk when estimates were based on market-sampled crops, 23% appeared at risk when estimates were based on the HarvestPlus FCT, and 16% appeared at risk when estimates were based on samples from household farms. CONCLUSION: The use of FCTs to calculate Zn intake overestimated the risk of dietary inadequacy for children who primarily consumed staple crops that were produced on household farms, but underestimated the risk for children who primarily consumed staple crops that were purchased at market. More information on the Zn content of staple crops in developing countries could lead to more accurate estimates of dietary intake and associated deficiencies.

Nitrogen speciation and transformations in fire-derived organic matter.

Vegetation fires are known to have broad geochemical effects on carbon (C) cycles in the Earth system, yet limited information is available for nitrogen (N). In this study, we evaluated how charring organic matter (OM) to pyrogenic OM (PyOM) altered the N molecular structure and affected subsequent C and N mineralization. Nitrogen near-edge X-ray absorption fine structure (NEXAFS) of uncharred OM, PyOM, PyOM toluene extract, and PyOM after toluene extraction were used to predict PyOM-C and -N mineralization potentials. PyOM was produced from three different plants (e.g. Maize-Zea mays L.; Ryegrass-Lollium perenne L.; and Willow-Salix viminalix L.) each with varying initial N contents at three pyrolysis temperatures (350, 500 and 700 °C). Mineralization of C and N was measured from incubations of uncharred OM and PyOM in a sand matrix for 256 days at 30 °C. As pyrolysis temperature increased from 350 to 700 °C, aromatic C[bond, double bond]N in 6-membered rings (putative) increased threefold. Aromatic C[bond, double bond]N in 6-membered oxygenated ring increased sevenfold, and quaternary aromatic N doubled. Initial uncharred OM-N content was positively correlated with the proportion of heterocyclic aromatic N in PyOM (R2 = 0.44; P < 0.0001; n = 42). A 55% increase of aromatic N heterocycles at high OM-N content, when compared to low OM-N content, suggests that higher concentrations of N favor the incorporation of N atoms into aromatic structures by overcoming the energy barrier associated with the electronic and atomic configuration of the C structure. A ten-fold increase of aromatic C[bond, double bond]N in 6-membered rings (putative) in PyOM (as proportion of all PyOM-N) decreased C mineralization by 87%, whereas total N contents and C:N ratios of PyOM had no effects on C mineralization of PyOM-C for both pyrolysis temperatures (for PyOM-350 °C, R2 = 0.15; P < 0.27; for PyOM-700 °C, R2 = 0.22; P < 0.21). Oxidized aromatic N in PyOM toluene extracts correlated with higher C mineralization, whereas aromatic N in 6-membered heterocycles correlated with reduced C mineralization (R2 = 0.56; P = 0.001; n = 100). Similarly, aromatic N in 6-membered heterocycles in PyOM remaining after toluene extraction reduced PyOM-C mineralization (R2 = 0.49; P = 0.0006; n = 100). PyOM-C mineralization increased when N atoms were located at the edge of the C network in the form of oxidized N functionalities or when more N was found in PyOM toluene extracts and was more accessible to microbial oxidation. These results confirm the hypothesis that C persistence of fire-derived OM is significantly affected by its molecular N structure and the presented quantitative structure-activity relationship can be utilized for predictive modeling purposes.

Sequential Ammonia and Carbon Dioxide Adsorption on Pyrolyzed Biomass to Recover Waste Stream Nutrients.

The amine-rich surfaces of pyrolyzed human solid waste (py-HSW) can be "primed" or "regenerated" with carbon dioxide (CO2) to enhance their adsorption of ammonia (NH3) for use as a soil amendment. To better understand the mechanism by which CO2 exposure facilitates NH3 adsorption to py-HSW, we artificially enriched a model sorbent, pyrolyzed, oxidized wood (py-ox wood) with amine functional groups through exposure to NH3. We then exposed these N-enriched materials to CO2 and then resorbed NH3. The high heat of CO2 adsorption (Q st) on py-HSW, 49 kJ mol-1, at low surface coverage, 0.4 mmol CO2 g-1, showed that the naturally occurring N compounds in py-HSW have a high affinity for CO2. The Q st of CO2 on py-ox wood also increased after exposure to NH3, reaching 50 kJ mol-1 at 0.7 mmol CO2 g-1, demonstrating that the incorporation of N-rich functional groups by NH3 adsorption is favorable for CO2 uptake. Adsorption kinetics of py-ox wood revealed continued, albeit diminishing NH3 uptake after each CO2 treatment, averaging 5.9 mmol NH3 g-1 for the first NH3 exposure event and 3.5 and 2.9 mmol NH3 g-1 for the second and third; the electrophilic character of CO2 serves as a Lewis acid, enhancing surface affinity for NH3 uptake. Furthermore, penetration of 15NH3 and 13CO2 measured by NanoSIMS reached over 7 µm deep into both materials, explaining the large NH3 capture. We expected similar NH3 uptake in py-HSW sorbed with CO2 and py-ox wood because both materials, py-HSW and py-ox wood sorbed with NH3, had similar N contents and similarly high CO2 uptake. Yet NH3 sorption in py-HSW was unexpectedly low, apparently from potassium (K) bicarbonate precipitation, reducing interactions between NH3 and sorbed CO2; 2-fold greater surface K in py-HSW was detected after exposure to CO2 and NH3 than before gas exposure. We show that amine-rich pyrolyzed waste materials have high CO2 affinity, which facilitates NH3 uptake. However, high ash contents as found in py-HSW hinder this mechanism.

Synergies between mycorrhizal fungi and soil microbial communities increase plant nitrogen acquisition.

Nitrogen availability often restricts primary productivity in terrestrial ecosystems. Arbuscular mycorrhizal fungi are ubiquitous symbionts of terrestrial plants and can improve plant nitrogen acquisition, but have a limited ability to access organic nitrogen. Although other soil biota mineralize organic nitrogen into bioavailable forms, they may simultaneously compete for nitrogen, with unknown consequences for plant nutrition. Here, we show that synergies between the mycorrhizal fungus Rhizophagus irregularis and soil microbial communities have a highly non-additive effect on nitrogen acquisition by the model grass Brachypodium distachyon. These multipartite microbial synergies result in a doubling of the nitrogen that mycorrhizal plants acquire from organic matter and a tenfold increase in nitrogen acquisition compared to non-mycorrhizal plants grown in the absence of soil microbial communities. This previously unquantified multipartite relationship may contribute to more than 70 Tg of annually assimilated plant nitrogen, thereby playing a critical role in global nutrient cycling and ecosystem function.

Fire-derived organic matter retains ammonia through covalent bond formation.

Fire-derived organic matter, often referred to as pyrogenic organic matter (PyOM), is present in the Earth's soil, sediment, atmosphere, and water. We investigated interactions of PyOM with ammonia (NH3) gas, which makes up much of the Earth's reactive nitrogen (N) pool. Here we show that PyOM's NH3 retention capacity under ambient conditions can exceed 180 mg N g-1 PyOM-carbon, resulting in a material with a higher N content than any unprocessed plant material and most animal manures. As PyOM is weathered, NH3 retention increases sixfold, with more than half of the N retained through chemisorption rather than physisorption. Near-edge X-ray absorption fine structure and nuclear magnetic resonance spectroscopy reveal that a variety of covalent bonds form between NH3-N and PyOM, more than 10% of which contained heterocyclic structures. We estimate that through these mechanisms soil PyOM stocks could retain more than 600-fold annual NH3 emissions from agriculture, exerting an important control on global N cycling.

Adsorption and desorption of ammonium by maple wood biochar as a function of oxidation and pH.

The objective of this work was to investigate the retention mechanisms of ammonium in aqueous solution by using progressively oxidized maple wood biochar at different pH values. Hydrogen peroxide was used to oxidize the biochar to pH values ranging from 8.1 to 3.7, with one set being adjusted to a pH of 7 afterwards. Oxidizing the biochars at their lowered pH did not increase their ability to adsorb ammonium. However, neutralizing the oxygen-containing surface functional groups on oxidized biochar to pH 7 increased ammonia adsorption two to three-fold for biochars originally at pH 3.7-6, but did not change adsorption of biochars oxidized to pH 7 and above. The adsorption characteristics of ammonium are well described by the Freundlich equation. Adsorption was not fully reversible in water, and less than 27% ammonium was desorbed in water in two consecutive steps than previously adsorbed, for biochars with a pH below 7, irrespective of oxidation. Recovery using an extraction with 2M KCl increased from 34% to 99% of ammonium undesorbed by both preceding water extractions with increasing oxidation, largely irrespective of pH adjustment. Unrecovered ammonium in all extractions and residual biochar was negligible at high oxidation, but increased to 39% of initially adsorbed amounts at high pH, likely due to low amounts adsorbed and possible ammonia volatilization losses.