RHIZOBIAL CYCLING BETWEEN DIVERSE LEGUMES IN CROP ROTATIONS: UNDERSTANDING THE EFFECTS ON SOIL AND PLANT HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028247
• Grant No.: 2022-67013-36673
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2021-11038
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• Program Code: [A1402]- Agricultural Microbiomes in Plant Systems and Natural Resources

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

Performing Department
Plant Science

Non Technical Summary
Farmers choose which cover crops to use because of the specific agroecosystem services they provide (e.g., Nitrogen management, nutrient scavenging, weed suppression, and pest reduction). Legumes are used as cover crops because they increase soil Nitrogen levels for future crops. Legumes accomplish this feat by establishing beneficial associations with rhizobial bacteria who perform biological nitrogen fixation and thus provide Nitrogen (N) fertilization without the economic and greenhouse gas emission costs of chemical N inputs. In addition to this direct effect, growing legume cover crops changes soil microbiomes via enrichment of specific strains, species, or genera of rhizobia. Some legume cover crop species are particular about which rhizobia they associate with, while others are generalists. Further, some cover crop species share rhizobial partners with future legume cash crops in rotational contexts, while others do not. Here field and greenhouse experiments will be used to test the hypothesis that rhizobial cycling and effective N- fixation depends on the specificity and degree of symbiotic partner overlap between cover crop and focal crop hosts. Sequencing data will be used to test methods that simultaneously examine species composition and strain-level variation and thus enable the examination of agricultural microbiome structure and functioning across scales. Ultimately, leveraging the power of host-microbiome manipulation to enrich specific populations 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	1644	1080	30%
102	4010	1100	30%
202	1820	1070	10%
202	1410	1070	10%
202	1414	1070	10%
202	1640	1070	10%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 202 - Plant Genetic Resources;

Subject Of Investigation
1410 - Beans (dry); 1644 - Winter annual legumes; 1640 - Alfalfa; 1820 - Soybean; 1414 - Lentil; 4010 - Bacteria;

Field Of Science
1070 - Ecology; 1100 - Bacteriology; 1080 - Genetics;

Keywords

n-fixation

legacy effects

legume

nodule microbiomes

partner-specificity

rhizobia

sequence-capture

Goals / Objectives
The goalofthis proposal is to explorethe possibility of leveraging legume cover crops to enrich soil microbiomes for specific rhizobia communities that benefit cash crops and increase soil health.In particular, this proposal will test the hypothesis that rhizobial cycling and effective N-fixation depends on the specificity and degree of symbiotic partner overlap between cover crop and focal crops.Aim 1 and 2 will leverage an existing cover crop mixtures field experiment to measure rhizobia sharing between legume cover crops and focal cash crops. The greenhouse experiment in Aim 3 will test a broader panel of cover cropand cash crop species.Aim 1:Measure nodule and rhizosphere microbiomes of legume cover crops grown singly and in mixtures across multiple years in a long-term field experimentNodule and rhizosphere sampling (Fall and Spring 2021-2024)Extract & Seq. Samples (Yearly)Analysis & Writing (2024)Aim 2: Test for legacies of cover crops on soybean nodule and rhizosphere microbiomesNodule and rhizosphere sampling (Early Fall 2022 and 2023)Conduct Competition Experiment (Winter 2023)Analysis & Writing (2024)Aim 3: Measure the effect of cover crop microbiomes on legume cash crop and soil healthFinalize plant species identities (Early 2022)Conduct Greenhouse Experiment (2023)Analysis & Writing (2024)

Project Methods
Aim 1: Measure nodule and rhizosphere microbiomes of legume cover crops grown singly and in mixtures across multiple years in a long-term field experimentEach fall and spring for three years (including the year before the start date of this grant), we will randomly sample nodule and rhizosphere microbiomes from eight crimson clover and eight field pea plants in each of four replicate plots. We will collect samples from treatments where each species is grown alone and together in a mixture of three or six cover crop species (Table 3). After vortexing the root systems in PBS buffer to collect microbes adhering to roots, we will dissect nodules from plants, pool them together, surface sterilize in 10% bleach, homogenize and enrich them for microbes via differential centrifugation. After DNA extraction, we will submit microbiome samples for 16S rRNA Amplicon sequencing (using standard lab methods). Because we expect nodule microbiomes to be far less diverse, we will also submit nodule samples for high coverage, whole-genome sequencing. Using this data, we will determine 1) What microbes inhabit nodules and rhizospheres of these two legume cover crops in the field. 2) Are nodule and rhizosphere microbiomes robust to being grown with additional cover crops? And 3) Are microbiome enrichments stable across seasonal and yearly environmental variation? If we find that microbiome differentiation persists despite additional cover crop species and seasonal and yearly environmental variation, it suggests that legume cover crop species could be a valuable and practical on-farm microbiome manipulation tool.Aim 2: Test for legacies of cover crops on soybean nodule and rhizosphere microbiomesEach fall for three years, we will sample nodule and rhizosphere microbiomes from eight soybean plants in each of four replicate plots conditioned by six different cover crop treatments (Table 3). We will process these samples in the same way as Aim 1. By comparing microbiome composition and diversity in the treatments containing legumes to the Triticale-only and no cover crop treatment, we will test if prior cover crops alter Soybean nodule and rhizosphere microbiome composition. By comparing the monoculture cover crop treatments, we will test if these effects are species-specific. By comparing the three and six species mixtures to the species alone, we will test if these effects are robust to the presence of additional cover crops. Finally, by comparing years, we will assess if these effects are stable across environmental variation. We expect to find a strong presence of Bradyrhizobium species that are the canonical inhabitants of Soybean nodules. However, a recent microbiome study revealed that Ensifer fredii becomes the primary nodule inhabitant in certain soil types so we are also ready to be surprised.Aim 3: Measure the effect of cover crop microbiomes on legume cash crop and soil healthIn this aim, we will test if nodule or rhizosphere microbiomes enhance focal crop growth if there is partner overlap between the cover and cash crop. Rather paradoxically--cash crop species that are generalists may be best placed to benefit from cover crop enrichment. Hosts that are selective already find partners in crowded microbiome communities. Thus, less selective hosts may by more sensitive to the frequency of rhizobial species in the soil. Here we use a greenhouse microbiome legacy experiment to test the effect of nodule and rhizosphere microbiomes isolated from five cover crops species (three specialists and two generalists; Table 1) on the growth and N-fixation of four focal cash crops (two generalists and two specialists, Table 2). I chose these species because they 1) are grown as a cash or cover crops in the US, 2) are included in the long- term cover crop mixtures experiment, 3) literature indicates partner preferences and overlap, and 4) they are amenable to greenhouse growth. The experiment is similar to my prior legacy experiment, and consist of two plant generations. The first generation of cover crops--grown in field soil--will create the enriched nodule and rhizosphere microbiome communities that we will use to inoculate focal crops in the second generation. We will use 16S rRNA amplicon and whole-genome sequencing (as in Aim 1 and Aim 2) to characterize the microbiome communities enriched by each cover crop species. To measure the functional consequences of cover crop nodule and rhizosphere microbiomes on cash crops, we will compare standard plant traits (above/belowground biomass, C: N ratio, chlorophyll content, nodulation traits) and soil health metrics. To create a baseline for plant microbiome effects, we will grow each focal species in a field soil microbiome with no history of plant enrichment. Lastly, to assess plant growth sans microbiome, we will grow each species in sterilized field soil.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Scientists at twoacademic conferences, K-12 students at the Women in Science outreach event (WISE), Attendees of Pennsylvania State University AgriculturalProgress Days Changes/Problems:I apologizefor this report's lateness. I thought I hit submit long ago. I only summarized here what happened during the reporting window from January 2023 toDecember2023. I have a fewminor additions to the project toreport, one of which is that this project also supportedthehonors thesis work of two undergraduatestudents. One studentconducted a small project studying the effect of domestication onPhaseolus(one of our legume cash crops)on rhizobia that we used as a pilot for the bigger experiment in Aim 3. The other student added measurements of caterpillar growth ratesofthree of our cash crops in the Aim 3 experiment, which tested if there is a cascading effectof past cover crops on herbivores. Secondly, As we have seen variability across the years in the preliminary sequencing data, we extended the sampling of soybeans for an additional year during this no-cost extension. I think it will strengthen the project outcomes, but I didn't originally propose it due to the short length of the seed grant. What opportunities for training and professional development has the project provided?During the reporting period, this grant supported training the following individuals in 1) field sampling techniques and 2) laboratory processing techniques, including DNA extractions, 3) manipulative greenhouse experiments, and 4) basic bioinformatics pipelines. One lab technician (1 URM) One postdoctoral scholar Twograduate students (2 URM) Three undergraduate students (3 URM) This project also supported one graduate student's presentation of results at a national conference and an undergraduate to begin her honors thesis on , which enhanced her training and professional preparation. How have the results been disseminated to communities of interest?One postergiven by a graduate student trainee would usethis system to try out a new microbial tracking method on cover crops. Funding for this two-year seed project was received five months earlier than requested, and thus, the start needed to be aligned with the experimental schedule, which began in September 2022. Thus, during this reporting period, we still needed to be at the point of reporting results or conducting outreach that included outcomes or results to target audiences. We will get our first major results in Spring 2024 (all sequencing from Aims 1 and 2 and experimental results from Aim 3 Phase II experiment). At least five students will be reporting on these results at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?During the no-cost extension fromJan 1st, 2024- Dec 31st 2024, we plan to: Aim 1:Measure nodule and rhizosphere microbiomes of legume cover crops grown singly and in mixtures across multiple years in a long-term field experiment Finish final timepoint of cover crop nodule and bulksoil sampling (Spring 2024) Extract DNA from remaining samples and send for sequencing Move forwardsequence analysis (graduate student and postdoc)and present at Evolution meeting in Montreal. Thesis chapter and manuscript drafting (led by lab PhDstudent who is a USDA predoctoral fellow) Aim 2: Test for legacies of cover crops on soybean nodule and rhizosphere microbiomes Conduct final sampling timepoint of nodule, bulk, and rhizosphere sampling of soybeans (Sept 2024) Extract DNA from finalsamples, amplify 16S, and conduct Amplicon sequencing. Analyze, write up, and present results for the first two years of sampling at the North American Symbiotic and Associative Nitrogen Fixation conference. Manuscript drafting and preparation (This aim is the firstchapter of asecond year graduate student's thesis). Aim 3: Measure the effect of cover crop microbiomes on legume cash crop and soil health Finish the second stage ofthe multigenerationgreenhouse cycling experiment, which started in Fall 2023, by growingsix cash crops with the nodule and rhizosphere microbiomesfromsix cover crop species. Harvest plants in late Spring 2024. Use as a training opportunity for data collection, analysis, and statistics fortwo undergraduate students, including an herbivory assay foran honors thesis. Use 16S amplicon sequencingto characterize the microbiome composition of the 96 inocula used for the cash crops and to assessthe nodule microbial composition in the cash crops. Analyze and present results at the North American Symbiotic and Associative Nitrogen Fixation conferenceand begin writing up (summer2024 onward).

Impacts
What was accomplished under these goals? During the reporting period fromJan 1st, 2023- to Dec 31st, 2023:we sampled the additional timepoints of an existing long-term cover crop mixtures field experiment to measure rhizobia sharing between legume cover crops and focal cash crops. We also conducted the first phase of the Aim 3 greenhouse experiment in Fall 2023, which will test a broader panel of cover crop and cash crop species and enable us to measure the plant benefits of rhizobial partner enrichment. The second half of this experiment will take place in the Spring of 2024. Aim 1:Measure nodule and rhizosphere microbiomes of legume cover crops grown singly and in mixtures across multiple years in a long-term field experiment Major activities completed / experiments conducted:Sampledcover crop nodules and bulksoil sampling (Spring/Fall 2023) Data collected:completed DNA extraction of samples to date and shotgun sequenced all 150 samples from cover crop nodule pools collected to date. Added anadditional diversity level to the sampling scheme and cover crops planted into fallow plots to measure baseline rhizobial diversity Analysis: Created preliminary bioinformatics pipeline from whole genome sequencing data for the 20 preliminary samples to prepare for downstream analysis of all samples. Key outcomes or other accomplishments realized: Two graduate student conference presentations and confirmation of successful sampling methodology. Aim 2: Test for legacies of cover crops on soybean nodule and rhizosphere microbiomes Major activities completed / experiments conducted: Nodule, rhizosphere, and bulk soil sampling--collected Fall 2023 cash crop samples as planned. Data collected: Ongoing DNA extraction &16S sequencing of samples. Summary statistics and discussion of results: none yet Key outcomes or other accomplishments realized: none yet. Aim 3: Measure the effect of cover crop microbiomes on legume cash crop and soil health Major activities completed / experiments conducted: Complete the first phase of the cover crop cycling experiment with six cover crop species. We will use those nodule and rhizosphere microbiomes to inoculate cash crops in Spring 2024 Data collected: Plant phenotypes, phenology, and a sample of microbiomes for 16S sequencing. Summary statistics and discussion of results: none yet Key outcomes or other accomplishments realized: none yet.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Jennifer E. Harris, Haneen Omari, Sohini Guha, Sharifa Crandall, Liana T. Burghardt, Estelle Couradeau. Exploring microbial habitat use across the soil, rhizosphere, and root continuum with BONCAT. Poster given at American Society of Plant Biologists Conference. Savannah, GA, August 2023.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Developed and taught guestlectureson 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 discussionon the use ofbeneficial microbes and an ethical case study on "Who owns the rights to Nitrogen-fixing corn?" Funding for this project was received five months earlierthan requested, and thus the start misaligned with the experimental schedule, which began in September 2022.Thus, we were not at the point during this reporting period to report results or conduct outreach that includes outcomes or resultstotarget audiences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the reporting period, this grant supported training the following individuals in 1) field sampling techniques and 2) laboratory processing techniques, including DNA extractions. Two lab technicians (2 URM) One postdoctoral scholar(1 URM) Three graduate students (2URM) Three undergraduate students (3URM) This project also supported one graduate student to present resultsat two national conferences that enhanced her training and professional preparation. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This proposal explores leveraging legume cover crops to enrich soil microbiomes for specific rhizobia communities that benefit cash crops and increase soil health.In particular, this proposal will test the hypothesis that rhizobial cycling and effective N-fixation depends on the specificity and degree of symbiotic partner overlap between cover crop and focal crops. During this reporting period, we sampled anexisting long-term cover crop mixtures field experiment to measure rhizobia sharing between legume cover crops and focal cash crops. We also conducted the preliminary background researchto enableus to start the Aim 3greenhouse experiment in Fall 2023, which will testa broader panel of cover cropand cash crop species and enable us tomeasure the plant benefits of rhizobial partner enrichment. Aim 1:Measure nodule and rhizosphere microbiomes of legume cover crops grown singly and in mixtures across multiple years in a long-term field experiment Major activities completed / experiments conducted: Ongoing cover crop nodule and rhizosphere sampling (planned Fall2021- Spring 2024) - completed Spring and Fall 2022 timepoints Data collected: Ongoing DNA extraction& sequencing ofsamples (Yearly) - completed extraction of samples to date andsequenced an initialsubset of 20 samples Summary statistics and discussion of results: Created bioinformatics pipeline to confirm rhizobial species inside cover crop nodules from whole genome sequencing data. Key outcomes or other accomplishments realized: Two graduate student conference presentations + generations of pipeline and confirmation of successful sampling methodology. Aim 2: Test for legacies of cover crops on soybean nodule and rhizosphere microbiomes Major activities completed / experiments conducted: Nodule and rhizosphere sampling (Early Fall 2022 and 2023) - Collected Fall 2022 timepoint as planned and completed monocultures and mixturesgreenhouse experiment (Spring 2022). Data collected:Ongoing DNA extraction& sequencing ofsamples. Summary statistics and discussion of results: none yet. Key outcomes or other accomplishments realized:none yet. Aim 3: Measure the effect of cover crop microbiomes on legume cash crop and soil health Major activities completed / experiments conducted: Finalize plant species identities (planned early 2022). Completed in Fall 2022 Data collected; none yet Summary statistics and discussion of results: none yet. Key outcomes or other accomplishments realized:none yet.

Publications

• Type: Journal Articles Status: Awaiting Publication 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: Published Year Published: 2022 Citation: Jennifer E. Harris, Regina B. Bledsoe, Haneen Omari, Sharifa Crandall, Liana T. Burghardt, and Estelle Couradeau. Do Plants Increase Microbial Activity in the Soil? Poster Presentation. Changing Microbiomes Symposium. June 2022. Boalsburg, Pennsylvania,USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Jennifer E. Harris, Regina B. Bledsoe, Haneen Omari, Sharifa Crandall, Liana T. Burghardt, and Estelle Couradeau. Do Plants Increase Microbial Activity in the Soil? Poster Presentation. Evolution Meeting. June 2022. Cleveland, Ohio, USA.