Source: WASHINGTON STATE UNIVERSITY submitted to NRP
FUNCTIONAL BIODIVERSITY OF SOIL NITROGEN-FIXING MICROBIAL COMMUNITIES ACROSS DIVERSE CROPPING SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030783
Grant No.
2023-67019-40214
Cumulative Award Amt.
$849,135.00
Proposal No.
2022-11099
Multistate No.
(N/A)
Project Start Date
Aug 15, 2023
Project End Date
Aug 14, 2026
Grant Year
2023
Program Code
[A1402]- Agricultural Microbiomes in Plant Systems and Natural Resources
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
This project aims to enhance agricultural productivity, sustainability, and resilience by increasing our understanding of nitrogen fixation by soil microbes. Despite its potential benefits, we still know relatively little about the diversity and abundance of nitrogen-fixing bacteria communities in the plant and soil microbiome and agricultural systems. To address this knowledge gap, we will conduct three closely related objectives. First, we will analyze soil samples collected by the national Soil Health Institute and the regional Washington Soil Health Initiative to survey the diversity and abundance of nitrogenous enzymes in the samples. We will also examine existing metadata on soil properties, cropping systems, and economic management to see how they relate to nitrogen fixation. Second, we will expand the development of NfixDB, a database being developed by our project team, to include agriculturally relevant sequences. We will use this database to design new primers to survey alternative nitrogenases that rely on different metal cofactors such as molybdenum, vanadium, and iron. We will then analyze the phylogenetic and biogeographic diversity of nitrogenase enzymes in relation to agricultural systems, particularly noting whether there are nitrogen-fixing bacteria that are unique to specific regions.Finally, we will conduct wheat experiments under controlled conditions to test whether metal cofactors limit the abundance and diversity of nitrogen-fixing bacteria communities. By doing this work, we hope to expand our knowledge of biological nitrogen fixation and find ways to manage agricultural systems more effectively.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1025210100035%
2014099104035%
2050199106020%
1330110107010%
Knowledge Area
133 - Pollution Prevention and Mitigation; 102 - Soil, Plant, Water, Nutrient Relationships; 205 - Plant Management Systems; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
4099 - Microorganisms, general/other; 0110 - Soil; 0199 - Soil and land, general; 5210 - Fertilizers;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1070 - Ecology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The primary goal of this research is to characterize free-living N fixer biodiversity and phylogenetics and determine interrelationships between genetics, the environment, and management that control N fixation across various agroecosystems. We propose to mine Soil Health Institute metagenomic data and other burgeoning public genomic and metagenomic databases to further develop our novel database (NFixDB). This database has been constructed using whole nitrogenase operons from high-quality complete genomes from the Genome Taxonomy Database (GTDB; further details below). NFixDB will form the platform for a suite of lab and greenhouse trials as well as both local and national biogeographic surveys to synthesize an integrative understanding of diazotroph diversity from genetic, taxonomic, and functional perspectives across agroecosystems and management regimes. Finally, we will test relationships between micronutrients, isozyme diversity, and N-fixation rates under controlled conditions. Together, these approaches will intersect to provide an integrated view of the biogeographical and anthropogenic drivers of soil diazotroph phylogeny, diversity, and function through the following objectives:Objective 1: Elucidate national and regional biogeographic patterns of soil diazotroph phylogenetic and functional diversity and identify environmental and phylogenetic drivers.Objective 2: Characterize phylogenetic diversity and novel taxa of free-living diazotrophs and the phylogenetic relationships between nif and alternative nitrogenases (vnf and anf).Objective 3: Link environmental and genetic controls on N-fixation potential to nitrogen-fixing communities.
Project Methods
Efforts:Collect soil samples and obtain 16S amplicon data and metadata from SHICollect soil samples and obtain soil nutrient and 16S amplicon data from WaSHISoil DNA extractionSoil nif high throughput SmartChip qPCR experiment and nif abundance data analysisSoil nif sequencing and diversity data analysisDatabase developmentPrimer designPhylogenetic analysisEndemism (geographic restriction of certain taxa)WA and MI field soil samplingGnotocosm experiment executionGreenhouse experiment executionGnotocosm & Greenhouse experiment soil and plant analysisConference presentationManuscript preparationEvaluation:The output of the project will be evaluated and quantified for its impact on the intended audience through a variety of evaluation studies. Key milestones and measurable or quantitative indicators of success will be identified, such as the number of students who participate in education and training opportunities, the number of publications or presentations resulting from the project, and the number of farmers or policy makers who use the project's findings to inform their management decisions. Data will be collected through surveys, interviews, and other methods to evaluate the impact of the project on the target audience(s). The evaluation plan will be used to continually assess the effectiveness of the project and make improvements as needed.

Progress 08/15/23 to 08/14/24

Outputs
Target Audience:The target audience of this project is researchers, farmers, conservation districts, other land managers, and the public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for 4 postdocs (2 WSU, 1 UNCC, 1 MSU), 2 PhD students (UNCC), and 5 undergraduates (2 WSU, 2 UNCC, 1 MSU). How have the results been disseminated to communities of interest?In addition to the 3 bioinformatics publications, project participants have given the following presentations related to this project: Soil Health Coffee Hour "Nitrogen fixing bacteria in wheat-based cropping systems" Kaviraj Singh Feb 28, 2024 Kaviraj Singh "Nitrogen fixing bacteria in wheat-based cropping systems" Washington Oilseed Commission meeting Feb 23, 2024 Kaviraj Singh "Nitrogen fixing bacteria in wheat-based cropping systems" Washington Grain Commission meeting Feb 13, 2024 Abby Grieb, Lisa Tiemann, Sarah Lebeis "Nitrogen dynamics in perennial bioenergy crop rhizospheres" GLBRC Annual Science Meeting, Lake Geneva, WI, May 13-15, 2024. What do you plan to do during the next reporting period to accomplish the goals?The next steps include completing DNA extractions and nif sequencing on the 245 WaSHI samples, as well as completing nifH sequencing on the amplicons from prairie sites and Cook Farm. This fall (2024) we will travel to Oregon State University to subsample soils from the Soil Health Institute's (SHI) collection encompassing samples from diverse cropping and management systems across North America. We expect to obtain 500 of these samples for DNA extraction. Nitrogenase diversity will begin by PCR amplification of the nifH nitrogenase gene from soil samples. We have the IGK3/DVV (Gaby et al., 2012) primers and will use those for PCR amplification. We will perform barcoding and amplicon library prep for Illumina Miseq sequencing at Michigan State University to obtain nifH sequence information for soil microbial communities. Resulting reads will be quality filtered and processed using the dada2 pipeline and assigned taxonomy using NFixDB (Bellanger, 2024). DeGenPrime-Ez, a GUI for DeGenPrime for ease of use primer design, also with features to extend access to color-blind and vision impaired individuals. DeGenPrime-Web, a website for ease of use primer design, also with features to extend access to color-blind and vision impaired individuals. FunGene2.0, a universal functional gene database featuring easy to use HMMs. Therion, universal tool for functional gene amplicon analysis featuring FunGene2.0 and NFixDB. Wet lab testing of novel nif primers will be carried out. Characterization of N-fixing isolates will continue, and gnotocosm experiments will be initiated.

Impacts
What was accomplished under these goals? We subsampled 245 soil samples from the Washington Soil Health Initiative's (WaSHI) collection. These soil samples were originally taken from different crop types across diverse regions in Washington state. Soil subsampling took place at WSU's Northwestern Washington Research and Extension Center in Mount Vernon, WA, where the soil samples were stored. Around one gram of soil was taken for each subsample. Soil subsamples are currently stored in sterile tubes at -80°C. Soil DNA extraction is currently ongoing. The table below describes the crop types and counties associated with subsamples. We processed a total of 192 soil samples for nifH sequencing from multiple agricultural and prairie sites near Pullman, WA. The prairie sites sampled were smoot, Kramer, Paradise, Paradise (old field CRP), and Whelan. The agricultural field sites were Cook LTAR west (business as usual) and east (no-till). DNA extraction: Soil DNA was extracted from each sample using the Kingfisher semi-automated platform and the Earth Microbiome Project high throughput DNA extraction protocol. nifH gene amplification: For analyzing diazotrophic community composition, nifH functional gene was amplified using the IGK3 (5′ -GCIWTHTAYGGIAARGGIGGIATHGGIAA-3′) forward primer and DVV (5′ -ATIGCRAAICCICCRCAIACIACRTC-3′) reverse primer, an ideal combination of primers to capture the greatest variety of diazotrophs (Gaby and Buckley 2012). PCR amplification of nifH was carried out in 15 μl reaction with 2 μl of DNA, 1× AmpliTaq Gold 360 Master Mix, 10 µM of forward and reverse primer each. The PCR program consisted of an initial denaturation at 95C for 10 min, followed by 34 cycles of denaturation at 95C for 30 s, annealing at 58C for 30 s, extension at 72C for 30 s and final extension at 72C for 7 min. Amplification of the target gene was confirmed by gel electrophoresis using a 1.5% agarose gel, run at 100V for 45 minutes. For next steps in the project, the amplified samples will be sent for sequencing and will be further analyzed for nifH community composition and structure across the different sampled sites. Gel electrophoresis picture of nifH amplification on subset of samples. Three bioinformatic tools were developed: MerCat2: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from omics data. NFixDB (Nitrogen Fixation DataBase)--a comprehensive integrated database for robust 'omics analysis of diazotrophs. DeGenPrime provides robust primer design and optimization unlocking the biosphere Diazotroph isolation: We have ~164 isolates from the N-free media, and about half have been confirmed as N-fixers using acetylene reduction assay. The isolates all came from KBS LTER rhizosphere soils from the following treatments: T1 conventional ag, corn-soy-wheat T3 Reduced input corn/soy/wheat with cover crop T4 Biologically based corn/soy/wheat with cover crop (no chemical inputs) T5 poplar planted 2019 T7 early succession grassland CF coniferous forest SF mid-successional forest DF late successional deciduous forest Nitrogen free media

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Figueroa III, Jose L., et al. "MerCat2: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from omics data." Bioinformatics Advances 4.1 (2024): vbae061.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Fulghum, Bryan, Sophie H. Tanker, and Richard Allen White III. "DeGenPrime provides robust primer design and optimization unlocking the biosphere." Bioinformatics Advances 4.1 (2024): vbae044.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Bellanger, Madeline, Jose L. Figueroa III, Lisa Tiemann, Maren L. Friesen, and Richard Allen White Iii. "NF ix DB (Nitrogen Fixation DataBase)a comprehensive integrated database for robust omics analysis of diazotrophs." NAR Genomics and Bioinformatics 6, no. 2 (2024): lqae063.