Progress 04/01/24 to 03/31/25
Outputs
Target Audience: The long-term goal of this research is to provide sustainable and effective agricultural products to farmers that mitigate the need for synthetic fertilizers that are continually becoming more costly and impact profitability. In addition, current farming practices result in excess nitrogen application that contributes to field runoff, polluting wells, rivers and streams, creating 'dead zones' in the Mississippi Delta, and because the chemical approach to synthetic nitrogen production (I.e., the Haber-Bosch synthesis) is an energetically intensive process, the production of fertilizer reduces the net energy obtained from crops intended to be used as biofuels. The immediate audience for this research is the scientific community working to identify beneficial microbes to promote plant health and reduce the requirement for fertilizer inputs on the farm. Further, commercial interest has emerged as a result of the NIFA grant and is in the early stages of spawning new collaborations that can increase the impact of the work, and hopefully deliver solutions for agriculture in a timely fashion. Changes/Problems: We experienced standard delays associated with hiring and onboarding new team members. However, these challenges have been addressed, and current efforts are moving forward with strong momentum and steady progress toward project goals. What opportunities for training and professional development has the project provided? This project has directly trained three postdoctoral researchers, two graduate students as well as two technicians and two data scientists as well as an undergraduate student participating in an REU program. How have the results been disseminated to communities of interest? J Zhou, S Kambhampati, DK Allen: SIMPEL 2.0: Automating Untargeted Isotope Labeling Analysis and Pathway Elucidation, The 18th Annual Science Retreat of the Donald Danforth Plant Science Center Scientific Retreat, St. Louis, MO (June 2024; poster) J Zhou, JP Noel, DK Allen, S Kambhampati: SIMPEL 2.0: Automating Untargeted Isotope Labeling Analysis and Pathway Elucidation, The 26th International Symposium on Plant Lipids, Lincoln, NE (July 2024; poster) J Zhou, JP Noel, DK Allen, S Kambhampati: SIMPEL 2.0: Automating Untargeted Isotope Labeling Analysis and Pathway Elucidation, International Camelina Conference, Lincoln, NE (July 2024; poster) R Bart: Novel microbes and newly assembled syncoms to promote sustainable agriculture, International Phytobiomes Conference, St. Louis, MO (November 2024; talk) S Arnesen, W Ranney, F Guo, J Sumner, R Bart: Identifying diazotrophs from root rhizosphere using variable nifH primers, International Phytobiomes Conference, St. Louis, MO (November 2024; poster) S Morley, V Vanyo, A Bravo, DK Allen: The fats of life: Lipid transfer and carbon partitioning in the nutrient exchange between plants, AMF and rhizosphere bacteria. Gordon Research Conference on Plant Lipid Structure, Function and Metabolism, Pomona, CA (January 2025; poster) Co-PI Allen provided a seminar at the Department of Plant Sciences, University of Cambridge entitled, Isotope-Assisted Metabolic Flux Analysis: Understanding Spatial and Temporal Metabolism for Crop Design. Cambridge, UK June 2024 What do you plan to do during the next reporting period to accomplish the goals? Identify and characterize "helper" microbes (Aim 1): We will analyze our field 16S datasets using Spearman correlation and network-based approaches to identify ASVs that consistently co-occur with our nifH-positive isolates. These analyses will prioritize candidate "helper" ASVs based on abundance correlations and network centrality. Once identified, we will target field samples rich in these candidate ASVs for metagenomic sequencing and reconstruction of Metagenome-Assembled Genomes (MAGs). We will then search for close matches to these MAGs within our culture collection and publicly available resources to identify cultivable strains that resemble our predicted helpers. If necessary, we may also attempt bulk culturing approaches, though we currently view metagenomics-guided identification as more efficient and promising. Advance metabolomics and software development (Aim 3.1): We will complete analysis of our current D?O labeling dataset and prepare a manuscript based on these results. This work includes continued use and validation of SIMPEL 2.0 for untargeted metabolomics. We also aim to refine SIMPEL 2.0 for public release, with improvements to usability and documentation to support adoption by the broader research community. Integration of MS2-based analysis remains a possible enhancement depending on outcomes and available resources. Improve rhizosphere carbon tracing under varying nutrient conditions (Aim 3.2): We are extending our isotope labeling experiments to 16 hours to improve carbon enrichment in the rhizosphere, enabling better resolution of metabolite transfer into microbial communities. These experiments will continue under both nitrogen-rich and nitrogen-limited conditions to assess the effects of nutrient availability on carbon allocation and exudation. We will also compare how different microbial communities impact exudate composition, colonization, and plant growth, to better understand the feedback between root exudates, microbiome composition, and plant nutrient uptake.
Impacts
What was accomplished under these goals?
1.1: To identify beneficial bacterial communities with nitrogen-fixing potential, we expanded our previous qPCR-based screen for the nifH gene by evaluating multiple primer sets. We tested four nifH primer pairs across 156 bacterial isolates obtained from N-free media. Our results revealed substantial variation in detection capacity across primer pairs, underscoring the limitations of relying on a single primer set. While initial efforts identified three nifH-positive Paenibacillus strains, expanded screening detected six Paenibacillus and ten Clostridium strains harboring nifH, many of which fell into distinct phylogenetic subclades, suggesting functional diversity. To overcome limitations of primer-based detection and gain insight into the full nitrogen fixation potential, we initiated whole genome sequencing (WGS) on all culturable isolates. Thus far, 130 of 203 isolates have been successfully sequenced. WGS has confirmed the presence of nifH and additional core and accessory nitrogenase genes in most of the Paenibacillus strains, while Clostridium isolates showed more variable gene content. These results will guide the selection of candidate nitrogen-fixers for downstream functional assays. 1.2: We continued refining our co-occurrence network analysis pipeline to identify microbial associations contributing to stable community structures. New analytical tools, including permutation tests, Dirichlet mixture models, and empirical Bayesian changepoint analyses, were integrated into our framework. Using datasets from two field sites (2021 and 2022), we identified 41 conserved microbial communities, largely in the endosphere, that reproducibly corelate with plant phenotypes of interest, such as yield. Current efforts are focused on generating hypotheses linking these communities to function and leveraging WGS data to uncover mechanisms beyond taxonomic identity. 1.3: Building on previous metabolomics efforts, we are leveraging a complementary project that explores a novel labeling strategy aimed at increasing the number and resolution of biologically relevant, identifiable metabolites. This approach will help refine our analysis by focusing on meaningful metabolic shifts associated with microbial interactions. In addition, we have developed methods of searching for metabolites that correlate with specific microbes and this work is currently under revision at the journal Phytobiomes. We originally developed these methods using a relatively constrained dataset from a large mesocosm experiment. Having developed the methods, we will now apply similar methods to the field data. 2.1: We expanded our culture-based isolation pipeline to recover potential nitrogen-fixing bacteria from rhizosphere samples identified in Task 1.1. A total of 203 isolates were recovered on nitrogen-free media under aerobic and anaerobic conditions. Using four nifH primer sets, we detected six Paenibacillus and ten Clostridium isolates positive for nifH. To improve detection and assess genomic potential, we initiated whole genome sequencing (WGS), successfully sequencing 130 isolates to date. WGS confirmed the presence of core nitrogenase genes (nifH/K/D) and accessory genes in most Paenibacillus isolates, while Clostridium strains showed more variable gene content. Genome mining is ongoing to identify traits relevant to nitrogen fixation and plant growth promotion, including auxin production, ACC deaminase, and biofilm formation. 2.2: To validate nitrogen-fixing capacity, we initially adapted an acetylene reduction assay (ARA) using a known free-living diazotroph (Azotobacter vinelandii) to establish a reliable positive control. However, repeated attempts to detect acetylene reduction in our slow-growing Paenibacillus and Clostridium isolates under in vitro conditions were unsuccessful. This may be due to suboptimal growth environments or missing plant-derived signals required for full expression of nitrogen fixation traits. To address this, we shifted to a greenhouse assay to evaluate nitrogen-fixing potential. Maize hybrid plants were grown under four nitrogen fertilization regimes (100%, 50%, 25%, 0%) and treated with one of three microbial inoculants: (1) a synthetic community of five nifH-positive isolates (three Paenibacillus, two Clostridium), (2) Azotobacter vinelandii, or (3) a water-only control. Plants were grown for three weeks with regular re-inoculation to support microbial establishment and persistence. After 4 weeks, we collected shoot and root biomass, performed dry weight measurements, and acquired imaging data throughout the experiment for digital phenotyping using the PlantCV platform. Preliminary biomass data suggest no significant differences in growth metrics across treatments. However, colonization and persistence of microbial strains in the rhizosphere are still being evaluated through 16S amplicon sequencing. This analysis will determine whether inoculated strains successfully established in the maize root environment and whether colonization correlates with plant phenotype or nitrogen status. These results will guide refinement of inoculation strategies and support further testing of strain function in planta. 2.3: As described in 2.2, we assembled and tested a SynCom composed of five nifH-positive "doer" strains with predicted nitrogen-fixing potential. While this initial community focused on functional nitrogenase activity, efforts to identify complementary "helper" strains that may enhance colonization, persistence, or overall SynCom stability are ongoing. 3.1: Efforts using D2O to study germination in seeds inoculated with or without microbes continue. Current efforts include extended studies with an increased number of labeling time points to monitor germination and establish pseudo-steady state metabolism. Targeted and untargeted metabolomics, including inspection of the isotopologues is ongoing. This data is being used to further refine the software package that we published last year called SIMPEL. Through the untargeted analysis, we identified 10,680 metabolomic features, of which 1,312 were enriched metabolites. These were grouped into 456 active bins (i.e., metabolites that contained multiple isotopologues and indicated labeling). During this period, both SIMPEL 1.0 and 2.0 were actively maintained and updated to enhance performance and incorporate new functionalities. New features added include a function for validating the steady-state assumption, network visualization, pathway enrichment visualization, and various comparison plotting options. We are currently evaluating results from both analyses and plan to extend this refined approach to the bacterial treatments outlined in Aim 2. 3.2: Additional methods were considered to effectively provide 13C to plants and monitor the interaction between corn and microbes, mediated by root exudate. We compared ¹³C-glucose with ¹³CO2. Glucose was provided through the stem of corn plants by one of several means and regardless of approach, showed limited incorporation (14% 13C labeling in root sugars and 30% in leaves after 24 hours). Provision of ¹³CO? through the leaves with transport to the roots resulted in significantly higher incorporation: 66% labeling in sugars, organic acids in roots, 70% in leaves, and 8-20% in protein hydrolyzed amino acids, depending on the tissue. Additionally, 2-8% labeling was observed in rhizosphere protein hydrolysates, confirming movement of labeled carbon into the root-soil interactions. These results validate that our system effectively tracks the movement of photosynthetically derived carbon into plant tissues and exudates and as the provision of carbon as ¹³CO? is the natural form for a plant, our in planta labeling experiments were determined to be the most effective approach for further study.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
S Koley, P Jyoti, M Lingwan, M Wei, C Xu, KL Chu, RB Williams, AJ Koo, JJ Thelen, D Xu, DK Allen: Persistent fatty acid catabolism during plant oil synthesis . Cell Reports 44, 115492, April 22, 2025
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Progress 04/01/23 to 03/31/24
Outputs
Target Audience:The long-term goal of this research is to provide sustainable and effective agricultural products to farmers that mitigate the need for synthetic fertilizers that are continually becoming more costly and impact profitability. In addition, current farming practices result in excess nitrogen application that contributes to field runoff, polluting wells, rivers and streams, creating 'dead zones' in the Mississippi Delta, and because the chemical approach to synthetic nitrogen production (I.e., the Haber-Bosch synthesis) is an energetically intensive process, the production of fertilizer reduces the net energy obtained from crops intended to be used as biofuels. The immediate audience for this research is the scientific community working to identify beneficial microbes to promote plant health and reduce the requirement for fertilizer inputs on the farm. Further, commercial interest has emerged as a result of the NIFA grant and is in the early stages of spawning new collaborations that can increase the impact of the work, and hopefully deliver solutions for agriculture in a timely fashion. Changes/Problems:A postdoctoral scientist, Dr. Poonam Jyoti, successfully competed for and was awarded a prestigious Assistant Professor Faculty Fellowship throught INSPIRE program in India. While this is an excellent training outcome for our labs, NIFA, and the Danforth Center, it leaves a vacancy related to aspects of this work. Dr. Jyoti will continue to use her developed expertise related to plant and microbe interactions, metabolism, nutrient exchange, isotopic labeling and metabolic flux analysis in her new professorial role which will magnify our efforts to sustainably address global challenges such as synthetic fertilizer use. She left the lab approximately the end of May and a recent advertisement has identified a candidate to complete additional aspects of the grant. The new hire will join the grant and teams, pending visa and moving to the U.S. What opportunities for training and professional development has the project provided?This project has directly trained two postdoctoral researchers and a graduate student as well as two technicians and two data scientists. How have the results been disseminated to communities of interest?Results have been disseminated through publications and DDPSC community seminars. Presentations given to the broader scientific community as seminars and poster presentations are listed below: R Bart: The Future of Plant-Environment Interactions: Challenges and Opportunities in a Changing Climate, Banbury Conference, Lloyd Harbor, New York October 2023 D Allen: Use of stable isotopes to study plant metabolism and root exudation, DOE-EMSL Workshop on Stable Isotopes, Richland WA January 2024 P Jyoti, S Morley, S Koley: Big Ideas 3.0, Donald Danforth Plant Science Center, St. Louis MO August 2023 S Arnesen: Harnessing plant beneficial rhizosphere microbial communities to improve plant health, Fast Talks with University of Nebraska Students, Donald Danforth Plant Science Center, St. Louis MO March 2024 P Jyoti, KL Chu, S Koley, S Kambhampati, A Onyshchenko, I Moroenyane R Bart, DK Allen: Understanding root exudates using stable isotopes. The 17th Annual Science Retreat of the Donald Danforth Plant Science Center Scientific Retreat, St. Louis MO June 2023 J Zhou, S Kambhampati, DK Allen: SIMPEL 2.0: Automating Untargeted Isotope Labeling Analysis and Pathway Elucidation, The 18th Annual Science Retreat of the Donald Danforth Plant Science Center Scientific Retreat, St. Louis MO June 2024 What do you plan to do during the next reporting period to accomplish the goals?During the next period we will: Continue mining rhizosphere and endosphere samples for addition candidate N-fixing bacteria (Aim 1.1). Test version 2 of our pipeline for identification of candidate "helper" bacterial strains (Aim 1). Characterize our candidate N-fixing isolates and begin assembling and testing syncoms based on candidate ASVs identified in version 2 of our pipeline. Apply the developed ARA method and refine isotope labeling with 15N to more rigorously characterize microbes identified as growing in N-free media and that have nifH expression (Aim 2.2). Further refine our ability to isotopically label and measure exudates from plants (Aim 3.2). Pursue additional isotopic labeling experiments for untargeted labeled metabolomics analysis with SIMPEL 2.0, and refine the software tool to assess a second mass spectrometric dimension (i.e., MS2 measurement from tandem mass spectrometry). The increased information content from MS2, coupled with linkage to KEGG metabolic pathways database, will enable more confident identification of unlabeled metabolites (Aims 1&3).
Impacts
What was accomplished under these goals?
1.1: To identify beneficial bacterial communities that robustly colonize maize roots and potentially enhance plant performance we conducted a comprehensive analysis of field data focusing on the nifH gene, a marker for nitrogen-fixing bacteria. Using qPCR with nifH-specific primers, we screened 1502 samples from field experiments conducted over two seasons (2021 and 2022). Our analysis so far has prioritized the 2022 field data, encompassing 960 samples from 64 plots of 8 maize-teosinte introgression genotypes. From the 2022 dataset, we successfully screened 932 samples for nifH gene abundance, revealing significant variability in nifH abundance across different sample zones (endosphere, rhizosphere, soil) and field sections. nifH abundance correlated positively with certain soil properties (sulfur, soluble salts, soil pH) and negatively with zinc levels. We observed a positive correlation between nifH abundance and several plant phenotypes, including root dry weight, mid-season, kernel weight and flowering time, for at least one of the genotypes analyzed. Samples with high nifH abundance will be leveraged for isolating candidate nitrogen-fixing bacterial strains (Aim 2.1). 1.2: Our initial approach involved running co-occurrence and exclusion analyses to identify microbe-microbe associations that could contribute to the stability of synthetic communities (SynComs). Over the past year, we have compared multiple flavors of network analysis, to better understand microbial interactions. To date, we have not identified a "smoking gun" community that we would predict to significantly impact plant health. However, these computational approaches will continue in the next year. To enhance our analysis, we incorporated an additional year's dataset (2022) to serve as a training dataset, thus addressing concerns about false positives and reproducibility. Our focus shifted to exploring quality control metrics, particularly balancing presence and absence data, to refine our approach and manage the high levels of hits encountered. We evaluated existing R packages for network analysis and are modifying and developing our pipeline to better fit our specific needs. 1.3: Initial efforts focused on analyzing the 2021 dataset, considering target metabolites as phenotypes of interest. However, no strong patterns or relationships between microbial communities and plant phenotypes were observed. Multi-omics approaches, including Procrustes analysis and multiple coinertia analysis, were used to compare the principal components of the microbiome and metabolome, but neither method revealed significant relationships. We then expanded our analysis to include the 2022 dataset. Using XCMS for peak detection and retention time alignment, followed by salt cluster filtering and PQN normalization, we processed metabolomics data from root and shoot samples. Applying the Kruskal-Wallis test and Dunn's test, we identified significant differences in metabolite intensities: 1309 differential features in shoots and 104 in roots. Kendall's tau correlation analysis revealed significant correlations between specific metabolites and plant phenotypes, such as kernel weight, seed count, days to flowering, and plant height. Additionally, we identified significant correlations between metabolite levels and soil properties like phosphorus, potassium, zinc, and soil pH, particularly in shoots. Despite these efforts, no definitive patterns emerged linking microbial communities directly to root metabolic profiles. Feature identification is ongoing, and we are continuing to refine our methods. 2.1: We focused on isolating nitrogen-fixing bacteria from field samples identified as enriched in the nifH gene as part of task 1.1. Instead of using brute force or limiting dilution approaches, we opted for isolation on nitrogen-free (N-free) media to enhance the selection of nitrogen-fixing bacteria. A pipeline was developed for the isolation process. Field samples underwent heat treatment and serial dilutions were then plated on N-free media under both anaerobic and aerobic conditions. This method allowed us to isolate 156 bacterial strains from three field rhizosphere samples. Colonies were subsequently grown in rich media, and their identities were confirmed through 16S sequencing of the V4/V5 region and nifH PCR. The isolation approach leveraging N-free media proved effective, with most wells containing a single genus, indicating successful purification of isolates. Notably, we confirmed the presence of the nifH gene in three new bacterial isolates: Paenibacillus taohuashanense, Pseudomonas granadensis, and Paenibacillus helianthin. Nitrogen fixing ability with be confirmed in these strains and they will be further characterized using in vitro studies and seedling assays, as outlined in subsequent tasks. 2.2: Task 2.2 focuses on characterizing the candidate n-fixing bacteria isolated in Task 2.1, including validation and assessment of nitrogen-fixing abilities. We are adapting a previously established acetylene reduction assay (ARA) to measure the nitrogenase activity of these strains by converting acetylene to ethylene, quantified via gas chromatography. We are optimizing this assay with a known free living diazotroph. Preliminary results are encouraging, but further optimization is required to accurately measure nitrogenase activity in the new isolates (task 2.1). 2.3: Currently, we are still refining our method for identifying 'helper' strains, and therefore, no SynComs have been assembled or tested to date. 3.1: We performed D2O labeling on seeds germinated for 36 hours, comparing mock treatments with BLO402 (a bacterial strain previously shown to be capable of promoting plant growth). Using a qExactive Orbitrap mass spectrometer and our expanded SIMPEL 2.0 software, we conducted untargeted metabolomics analysis, identifying 36,152 metabolomic features, of which 2,770 were enriched metabolites. These grouped into approximately 900 active bins contributing to germinative metabolism. We are currently evaluating the metabolic response to BLO402, with plans to extend this analysis to SynComs from previous aims. 3.2: To study root exudates, we developed a new method to overcome the limitations of hydroponic and natural soil systems. Sterile seeds are grown in a sand-gravel mix for 10 days, followed by a water wash with an internal standard. The exudates are collected, centrifuged to pellet debris, desalted, and analyzed via mass spectrometry. This method, tested with multiple substrates and growth mediums, evaluated target exudates like glucose, phenylalanine, sucrose, serine, and leucine/isoleucine at 7, 10, and 12 days of growth. We found that root exudation peaks mid-day by collecting exudates at six time points over a 24-hour period. These findings show plants start exuding detectable amounts at the early seedling stage, with maximum exudation mid-day. For stable isotope-based labeling, we used 13CO2 to analyze root exudates focused on central metabolites. Initial experiments recovered labeled metabolites in exudates after 12 hours, with enrichment increasing over time. This method distinguishes root exudates from other rhizosphere metabolites. Approximately 20 labeled metabolites were identified after 12 hours of labeling, with similar levels at 24 hours, indicating saturation of leaf photosynthetic contribution relative to remaining seed-derived carbon. These advances in root exudate collection and stable isotope labeling, though still being refined, will enable detailed studies on plant-microbe interactions and potentially enable complementary experiments leveraging the DOE National Lab resources.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Kambhampati et al., 2024. SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism DOI: https://doi.org/10.1038/s42003-024-05844-z
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
P Jyoti, KL Chu, S Koley, S Kambhampati, A Onyshchenko, I Moroenyane R Bart, DK Allen: Understanding root exudates using stable isotopes. The 17th Annual Science Retreat of the Donald Danforth Plant Science Center Scientific Retreat (June 2023; poster)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
J Zhou, S Kambhampati, DK Allen: SIMPEL 2.0: Automating Untargeted Isotope Labeling Analysis and Pathway Elucidation, The 18th Annual Science Retreat of the Donald Danforth Plant Science Center Scientific Retreat (June 2024; poster)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
PI Bart presented this work at a Banbury conference entitled The Future of Plant-Environment Interactions: Challenges and Opportunities in a Changing Climate in October 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
S Arnesen: Harnessing plant beneficial rhizosphere microbial communities to improve plant health. Fast Talks with University of Nebraska Students (March 2024: Talk).
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