LAYING THE GROUNDWORK TO BREED FOR MUTUALISMS:TRACKING LONG-TERM ADAPTATION OF NITROGEN-FIXING RHIZOBIA TO ALFALFA IN AGRICULTURAL FIELDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028093
• Grant No.: 2022-67013-36860
• Cumulative Award Amt.: $650,000.00
• Proposal No.: 2021-09935
• Project Start Date: Feb 15, 2022
• Project End Date: Feb 14, 2026
• Grant Year: 2022
• Program Code: [A1102]- Foundational Knowledge of Agricultural Production Systems

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505

Performing Department
Plant Science

Non Technical Summary
As cash, forage, and cover crops, legumes play a crucial role in global nutrition and food security. Legumes establish beneficial associations with rhizobial bacteria who perform biological nitrogen fixation (BNF)--providing Nitrogen (N) fertilizer to plants without the economic and greenhouse gas emission costs of chemical N inputs. However, this mutualism is far from optimized in agricultural systems. Crop benefits are highly variable and context-dependent and existing inoculants often fail to survive or compete in agricultural soils. In the northeastern United States, the perennial forage and cover crop alfalfa is a critical component of conventional and organic agroecosystems. This proposal leverages existing alfalfa variety trials to assess the ability of alfalfa (Medicago sativa) to steer the evolution of their mutualistic partners in the face of environmental variation. By applying novel sequencing technologies previously validated in Medicago truncatula to field trials, we will measure the extent and genomic basis of rhizobial adaptation to alfalfa varieties across seasons and years. By conducting paired multi- and single-strain greenhouse experiments using isolates from the trial, we will assess strain competition and plant N-fixing benefits to enable screening for alfalfa varieties' ability to reward helpful rhizobia. This proposal will benefit 1) legume breeders by establishing a screening pipeline and 2) organic and conventional growers by contributing to the selection of legume varieties. Ultimately, leveraging legume hosts to select on existing rhizobia represents a novel, cost-effective avenue to promote efficient N-fixation, thereby supporting robust yields, improving soil health, and decreasing chemical N-inputs and greenhouse gas emissions.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	4010	1070	30%
203	1640	1080	30%
202	1640	1081	20%
206	1640	1060	20%

Knowledge Area
206 - Basic Plant Biology; 102 - Soil, Plant, Water, Nutrient Relationships; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
1640 - Alfalfa; 4010 - Bacteria;

Field Of Science
1060 - Biology (whole systems); 1070 - Ecology; 1081 - Breeding; 1080 - Genetics;

Keywords

alfalfa

biological nitrogen fixation

crop selection

plant yield

rhizobia

soil health

Goals / Objectives
The development of extensive genomic and bioinformatics resources in legume-rhizobium systems allows us to create novel sequence-based methods to answer fundamental questions about rhizobial fitness and the genomic basis of adaptation to abiotic and biotic contexts. We propose to apply these cutting-edge, lab-based methods to ongoing Pennsylvania State University variety trials of the perennial forage and cover crop alfalfa (Medicago sativa). This proposal will:Objective 1: Translate sequencing methodologies to the field to measure seasonal and alfalfa-mediated evolutionary processes in established varietal trialsObjective 2: Test a novel evolution-informed phenotyping pipeline to measure the ability of alfalfa varieties to reward helpful strainsObjective 3: Identify rhizobia candidate genes underlying: 1) adaptation to alfalfa nodules in agricultural fields, 2) competitive success across alfalfa varieties and environmental variation, and 3) N-fixation benefits to plantsIn particular, this proposal aims to assess the potential to select legume varieties that can maintain large populations of mutualistic rhizobia via rewards mechanisms. To accomplish this goal, we will employ interconnected field and greenhouse-based experiments. In Aim 1, we will measure seasonal- and host-mediated selection in alfalfa nodules in the variety trials over multiple years. In the greenhouse, we will assess the competitive strain fitness of field isolates (Aim 2) and quantify single-strain benefits (Aim 3).Aim 1:Measure seasonal- and variety-mediated selection on rhizobia in alfalfa variety trialsAim 2: Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variabilityAim 3: Use single-strain inoculations to test N-fixation benefits to each alfalfa variety

Project Methods
Aim 1 Overview:Measure seasonal- and variety- mediated selection on rhizobia in alfalfa variety trialsOur current method for assessing relative strain frequencies and fitness (Kessner et al., 2013) depends on a known initial strain mixture. When sampling nodules from fields, we will never have that luxury--there is just too much diversity. However, as long as we have a sufficient reference genome, we can measure shifts in individual variants between samples or across time to identify genes of interest via allele frequencies without knowinga prioriwhich strains are present.In the alfalfa variety trial, I propose to track selection onEnsifergenes in nodules of five alfalfa varieties to test for long-term adaption in PA fields (Figure 5). At each proposed sampling timepoint (Spring and Fall) and planting year (2016, 2017, 2020) and for each variety (5), we will unearth 8-12 plants and collect ~100 nodules. We will surface sterilize nodule pools, homogenize them in NaCl solution, and isolate rhizobia using a series of differential centrifugation steps that enrich undifferentiated bacteria (Burghardt et al., 2018). Pellets will be stored at -20°C until we extract DNA using the UltraClean Microbial DNA Isolation Kit (no. 12224; Mo Bio Laboratories). We will submit samples to either the PSU or UMN core genomics facility for Illumina library prep and ~ whole-genome sequencing using Illumina NovaSeq (125 bp paired-end reads to ~100X coverage). To identify putative regions under selection in the variety trials, we will identify allelic variants showing the strongest statistical signal of change in frequency between seasons, hosts, and plantings. As Burghardt et al. (2018) described, we will use a genome-wide scan for regions where allele frequencies change across contrasts. Briefly, each variant site will be tested using a generalized linear model with a binomial link and read counts supporting each allele as the response. By tracking allele frequency shifts in late-fall and late-spring for three years, we will be able to identify genes under repeated seasonal selection, genes underlying multi-year adaptation to alfalfa generally, and genes unique to specific alfalfa varieties (Figure 5a). Because of the wealth of information aboutEnsifergenomes we will immediately make functional inferences about critical selective pressures and home in on essential genome regions for downstream functional work.Aim 2 and 3 Overview:Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variabilityUse single-strain inoculations to test N-fixation benefits to each alfalfa varietyI will screen a new PennsylvaniaEnsiferstrain collection to identify alfalfa varieties that choose (Aim 2) and reward (Aim 3) the most beneficial strains. I will conduct Aim 2 across multiple environmental variables to gain an understanding of the sensitivity of competitive fitness to ecological variability. This greenhouse experiment will assess a new Pennsylvania soil and alfalfa selectedEnsiferstrain collection for strain competitive fitness across alfalfa varieties and environmental variability (10 replicates x 5 varieties x 4 environments). We will test the sensitively of competitive fitness in response to two agriculturally relevant environmental manipulations: the largely overlooked factor of rhizobial density in the soil (e.g., Yan et al., 2014) and the intensely studied aspect of Nitrogen addition (e.g., Heath et al., 2010; Regus et al., 2014, 2017; Bever, 2015; Weese et al., 2015; Grillo et al., 2016; Wendlandt et al., 2019; Zhang et al., 2020). Previously, we found inMedicago truncatulathat rhizobial density had a surprisingly strong host-dependent effect on strain competitive fitness. In contrast, the Nitrogen addition had a remarkably weak impact (Burghardt et al., 2020b). If confirmed in alfalfa, perhaps chemical N-addition in agriculture is not as terrible for N-fixing mutualisms that evolutionary theory suggests (Frederickson, 2017; Porter and Sachs, 2020).We will conduct our mixed inoculation experiment using the 120Ensiferisolates from the PSU alfalfa varietal trials. In brief, we will grow each of theEnsiferstrains individually in culture and then combine in equal volume to generate an inoculum with approximately equal representation from each of 120 strains (Figure 2). We will assess the composition of this initial inoculum via sequencing. Seeds of each alfalfa variety will be scarified, bleached, rinsed, stratified, and germinated on wet filter paper planted into pots filled with a sterilized mix of field soil, sand, and turface. Pots will be inoculated with ~105 (low density) or ~107 (high density) rhizobial cells of the mixed inoculant community. After plants establish, plants will be thinned to one per pot and will receive N-free fertilizer once per week and water as needed. N-addition plants will receive an increasing schedule of N-addition as they grow. Four months after planting, we will dissect roots, sample nodules from each plant. In addition to harvesting the nodules for strain frequency assessments, we will measure nodule number, nodule size, and vegetative and root biomass. Nodule pools will be processed and sequenced as in Aim 1. We will identify variants in genes with strong and consistent shifts in allele frequency among host varieties (as described in Aim 1). In addition, because this experiment starts with a known strain community, we will use the maximum-likelihood approach implemented in HARP (Kessner et al., 2013) to calculate strain frequencies and assess the relative importance of host varieties and environmental variation on strain diversity and strain competitive fitness. We will analyze these traits using Redundancy Analysis (vegan package Oskanen et al., 2017) and linear and multivariate methods in R (Development Core Team, 2016) as in our previous publications.A primary prediction from previous work is that the alfalfa variety will be the strongest driver of strain fitness variation followed by inoculum density and Nitrogen addition.Aim 3 methods, expected results, and analysis:We will perform a standard single-strain plant growth assay using fifteen competitive and fifteen noncompetitive strains from Aim 2 (30 strains x 5 varieties x 8 reps = 1200 plants). To assess individual level differences in controlled greenhouse environment, a single plant will be grown in each replicate pot in a complete randomized block design. All plants will receive the same amount of Nitrogen-free fertilizer (Barker et al., 2006) as needed. A mix of field soil, sand, and turface will be sterilized twice prior to seed planting and approximately 106 cells of one of Ensifer strain applied to each. In addition to assessing alfalfa biomass, we will also measure early growth rate, flowering time, and Chlorophyll A content in the leaves across multiple time points. After four months of growth, plants will be harvested and nodule size, morphology, and number assessed using standard image analysis protocols in image analysis software (e.g., ImageJ). Plant benefit data will be analyzed using standard generalized linear mixed models in R with an appropriate link for each trait. Models will include a random effect for replicate block to control for the experimental design. We expect only weak concordance between plant benefit proxies, and we expect strain identity to have large and variable benefits across alfalfa varieties. After calculating strain-specific benefits, we will test for an overall signal of host rewards and adaptation. Based on results from Medicago trucatula, we expect to find competitive strains to be more beneficial to hosts than noncompetitive strains. However, we expect for the strength of that relationship to vary across alfalfa varieties. Those with the strongest relationship have best potential to effectively manage rhizobial partners.?

Progress 02/15/23 to 02/14/24

Outputs
Target Audience: Scientists at two academic conferences (the International Society for Molecular Plant-Microbe Interactions and the American Society of Plant Biologists Conference) Members of the American Forage and Grassland Council who attend Ag Progress Days in Aug 2022 Developed and taught guest lectures on nitrogen-fixing symbiosis in Ag for undergraduate (two classes) and graduate students (two classes) in the College of Agriculture at Penn State University. Revised curriculum for the senior capstone class I teach, "Emerging Issues in the Plant Science", to include an expert panel discussion on using beneficial microbes and an ethical case study on "Who owns the rights to Nitrogen-fixing corn?" Middle school students participating in WISER outreach events Changes/Problems:I will submit the paperwork for a no-cost extension for this award in the coming months. We are making good progress, all things and COVID delays considered, but I received the funding six months earlier than requested, which didn't align with the constraints of ourfield sampling timeline. As of May 2024, we have most of the sequencing data. Now, we begin the challenging,exciting, and novel part of assembling genomes, tracking allele frequency shifts,making sense of the data, and designing the follow-up experiments. What opportunities for training and professional development has the project provided? Training of three undergraduate students (3 URM), two lab technicians, one postdoc (URM), and three graduate students (2 URM) in genuine field research and lab sample processing, including sampling and experimental design. Training of one undergraduate student (1 URM) and one lab technician in DNA extraction and sequencing submission processes. Training of one URM graduate student, two URM undergraduate students, and one lab technician on designing and conducting controlled experiments in the greenhouse One graduate student and one undergraduate presented scientific posters on strain collection and single strain phenotypesat two different conferences. How have the results been disseminated to communities of interest?We are still early in the data collection phase, but preliminary results were presented at two conferences in summer of 2023. What do you plan to do during the next reporting period to accomplish the goals?From Feb 15th, 2024 to Feb 14th, 2025 we will accomplish the following tasks: Aim 1: Measure seasonal- and variety-mediated selection on rhizobia in alfalfa variety trials In June and Nov 2024, continue collecting alfalfa nodule pools, rhizosphere, and bulk soil samples from 3 host varieties in the youngest 2022 established plot. Examine microbiome community composition across years and seasons in alfalfa rhizosphere using 16S from a subset of alfalfa samples to determine the best path forward for rhizosphere and bulk soil samples. Analyze and present phenotypic data from the field, including submitting soil samples for characterization. Analyze and develop methodologies to interpret sequencing data from the field. Present results at the North American Symbiotic Nitrogen Fixation meeting in June 2024 andthe New PhytologistNext Gen Scientists Conferencein June 2024, Aim 2: Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variability Analyze sequence data from nodule pools to infer the competitive fitness of 117 rhizobial strains from an experiment conducted in Spring 2023. Assuming our request for a no-cost extension is approved, these results will inform the design of a follow-up experiment on the environmental sensitivity of strains to be conducted in Spring 2025. Aim 3: Use single-strain inoculations to test N-fixation benefits of each alfalfa variety Analyze and present results from 1500-plant single-strain greenhouse experiment in Spring 2023 measuring both host and rhizobial benefits. Create denovo assemblies and map genomes to reference to enable gwas for symbiotic traits including lobing, nodule number, and plant benefit. Present results at the North American Symbiotic Nitrogen Fixation meeting in Burlington, VT,in June 2024 and the TriSocieties meeting in November 2024

Impacts
What was accomplished under these goals? From Feb 15th, 2023 to Feb 14th, 2024 we accomplished the following tasks: Aim 1: Measure seasonal- and variety-mediated selection on rhizobia in alfalfa variety trials In June and Nov 2023, alfalfa nodule pools, rhizosphere, and bulk soil samples were collected from 3-5 host varieties across multiple plots. Processed samples, extracted DNA, and sent all 270 pools collected to datefor whole-genome Illumina sequencing In Feb 2024 Quantified nodulation dynamics, nodule prevalence, and branching in the field and began analysis Aim 2: Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variability Created new strain collection and submitted 120 strainsfor PacBio long-read sequencing to enable Select and Resequence analysis,comparative genomics, and genome-wide associations studies Conducted preliminary competitive fitness experiment in Spring 2023, extracted DNA, and sent for sequencing with Aim1 samples. Aim 3: Use single-strain inoculations to test N-fixation benefits of each alfalfa variety Strain collection from Aim 2also enabled this aim. Successfully conducted 1500-plant single-strain greenhouse experiment Spring-Summer 2023, found massive variation in strain benefits provided to hosts from strains isolated from one alfalfa variety trial location. This gives us confidence in correlating rhizobial fitness Aim 2 with plant benefits in Aim 3 to identify hosts better at selecting beneficial rhizobia. We also measured standard symbiotic traits (e.g. nodule number). Beginning to conduct data analysis and presentresults.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Liana T Burghardt and George C diCenzo. The evolutionary ecology of rhizobia: multiple facets of competition before, during, and after symbiosis with legumes. Current Opinion in Microbiology. Volume 72,2023,102281(https://doi.org/10.1016/j.mib.2023.102281).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Gil-Polo, M. A. , Bingham, E., Bledsoe, R. A. , Guha, S. (The Pennsylvania State University), Sydow, P. , Paillan, E. , Burghardt, L. T. (Co-Author). (June 2023). "Do rhizobia with divergent morphologies co-isolated from Pennsylvania alfalfa fields alter host growth and strain competition?," International Society for Molecular Plant-Microbe Interactions, Providence, RI USA, Contributed.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Paillan, E. , Gil Polo, M. A., Sydow, P. , Bingham, E., Bledsoe, R. , Frye, G., Harris, J. , Sydow, P. , Guha, S. , Burghardt, L. T. (August 2023). "Examining Environmental and Genetic Drivers of Alfalfa Nodulation and Nodule Branching Over Time and Space," American Society of Plant Biologists, Savannah, GA, USA, Contributed.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Burghardt, L. T. , Guha, S., Bledsoe, R. A., Epstein, B., Heath, K., Young, N., Tiffin, P. "From genes to genera: Assessing genetic determinants of rhizobia fitness in legume nodules across scales," (August 2023). American Society of Plant Biologists, Savannah, GA, USA, Invited.

Progress 02/15/22 to 02/14/23

Outputs
Target Audience: Scientists at two academic conferences (the North American Symbiotic Nitrogen Fixation Conferenceand Changing Microbiomes Conference) Members of the American Forage and Grassland Council who attend Ag Progress Days inAug 2022 Developed and taught guest lectures on nitrogen-fixing symbiosis in Ag for undergraduate (two classes) and graduate students (two classes) in the College of Agriculture at Penn State University. Revised curriculum for the senior capstone class I teach, "Emerging Issues in the Plant Science", to include an expert panel discussion on usingbeneficial microbes and an ethical case study on "Who owns the rights to Nitrogen-fixing corn?" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training of three undergraduate students (3 URM), twolab technicians, one postdoc (URM), and three graduate students (2 URM) in genuine field research and lab sample processing,includingsampling and experimental design. Training of oneundergraduate student(1URM) and one lab technicianin DNA extraction and sequencing submission processes. Training of one URM graduate student, one URM undergraduate student, and one lab technicianin strain isolation protocols, as well as the use of MALDI-tof and Sanger Sequencing for strain identification. One graduate student presented her first two scientific posters on the creation of the strain collection at two conferences. How have the results been disseminated to communities of interest?Presentations at two scientific conferences: North American Symbiotic Nitrogen Fixation Conference, Madison, WI, June 2022 Changing Microbiomes Conference, Boalsburg, PA, May 2022 Outreach presentations in conjunction with Penn State Ag Progress Daysat the alfalfa variety trialplots to members of the Pennsylvania Chapter of the American Grassland and Forage Council What do you plan to do during the next reporting period to accomplish the goals?From Feb 15th, 2023to Feb 14th, 2024we willaccomplishthe following tasks: Aim 1:Measure seasonal- and variety-mediated selection on rhizobia in alfalfa variety trials In June and Nov 2023,continue collectingalfalfanodule pools, rhizosphere, and bulk soilsamples from 3-5 host varieties across multiple plots in two locations. Processsamples, extract DNA, and send ~ 270 remaining nodule samples forwhole genomesequencing. Quantify nodulation dynamics nodule prevalence, and lobing in the field Examine microbiome community composition across years and seasons in alfalfa rhizosphere using 16S from a subset of alfalfa samples to determine the best path forward to rhizosphere and bulk soil samples. Begin analyzing phenotypic data Aim 2: Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variability Conduct preliminary competitive fitness experiment with 117 rhizobial strainsin Spring 2023 Send initial communities and nodule pools for sequencing to inform the bigexperiment on the environmental sensitivity of strains to be conducted in Spring 2024. Aim 3: Use single-strain inoculations to test N-fixation benefits of each alfalfa variety Conduct a large, 1500-plant single-strain greenhouse experimentin Spring 2023 measuring both host and rhizobial benefits. Analyze and present results.

Impacts
What was accomplished under these goals? From Feb 15th, 2022 to Feb 14th, 2023 we accomplished the following tasks: Aim 1:Measure seasonal- and variety-mediated selection on rhizobia in alfalfa variety trials In June and Nov 2022 Collected alfalfanodule pools, rhizosphere, and bulk soilsamples from 3-5 host varieties across multiple plots in two locations. Processed samples, extracted DNA, and sent a subset for preliminary whole genomesequencing Aim 2: Screen rhizobia isolates for competitive fitness across alfalfa varieties and environmental variability Creatednew strain collection for use in this aim, including confirming identity as Sinorhizobium meliltoi via Sanger sequencing, creating replicate stocks, and extracting DNA for whole genome sequencing. Prepped for preliminary competitive fitness experiment to be conducted in Spring 2023 Aim 3: Use single-strain inoculations to test N-fixation benefits of each alfalfa variety Strain collection in Aim 2 also enabled this aim. Prepped for 1500 plant single-strain greenhouse experiment to be conducted in Spring 2023, including purchasing racks and pots, testing growth substrate, confirming germinationrhizobial growth, and plant phenotypingprotocols.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Gil Polo, M. A. (Author and Presenter - Graduate Student), Bledsoe, R. A. (Co-Author - Postdoctoral Student), Paillain, E. (Co-Author - Undergraduate Student), Rice, T., & Burghardt, L. T. (Co-Author). (June 2022). "Development of a Collection of Rhizobia Strains Adapted to Alfalfa in Pennsylvania Using High-Throughput Systems for Bacterial Cultivation and Identification," 25th North American Symbiotic Nitrogen Fixation Meeting, Madison, WI, USA, Contributed.
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Gil Polo, M. A. (Author and Presenter - Graduate Student), Bledsoe, R. A. (Co-Author - Postdoctoral Student), Paillain, E. (Co-Author - Undergraduate Student), Rice, T., & Burghardt, L. T. (Co-Author). (June 2022). "Development of a nitrogen-fixing bacteria collection from alfalfa variety trials to assess the evolution of microbial mutualists in agricultural ecosystems," Changing Microbiomes, Huck Microbiome Center, Boalsburg, PA, USA, Contributed.