Performing Department
(N/A)
Non Technical Summary
Mutualisms are a key biotic interaction that underlie the maintenance of biodiversity and multiple ecosystem functions in grazing lands. In particular, legume plants partner with bacteria called rhizobia in a close beneficial relationship. Rhizobia living within legume roots can obtain nitrogen (N) from the atmosphere and provide it to their legume hosts in a useable form, a method of obtaining N that most other plants do not have access to. The fixation of N from the atmosphere through the legume-rhizobia partnership comprises the largest natural source of N into grazing lands, promoting healthy forage production and soil health. Legumes are the third most diverse family of plants on Earth, with many legume species commonly found across agroecosystems. Yet, the vast diversity within this plant family is often overlooked, which can lead to inefficient or ineffectual management strategies in grazing systems. Legume species exist along a spectrum from specialists to generalists in their preferences for rhizobial partners. Specifically, some legume species are expected to be able to partner with a wide range of rhizobial bacteria, while others form more specific associations with just one or a few bacterial types. Theory and limited data indicate that these partnership preferences can have dramatic impacts on the ability of different legume species to grow together, but these patterns remain poorly characterized in grazing systems.In this Seed Grant, we aim to combine field and greenhouse data to understand the mechanisms driving legume-rhizobia coexistence in cattle pastures across North Carolina. The project team will include the Project Director, one graduate student, and two undergraduate students. We will first determine the rhizobial partners associated with five common species of pasture legumes across five North Carolina Department of Agriculture Field Stations. This will allow us to determine where these legume species exist along a gradient of specificity for rhizobial partners. Then we will determine whether legume specificity impacts the ability of these species to grow together and their landscape distributions. Finally, we will delve into the mechanisms underlying the legume distribution patterns we observe in the field through mechanism greenhouse studies. Results of this work will be disseminated to scientific audiences through peer-reviewed publications, as well as to shareholders at Field Days organized by the North Carolina Department of Agriculture Field Stations where the work will be conducted. Together, this information will identify key mechanisms driving legume species interactions and lay the groundwork for a future Full Proposal to USDA NIFA that explicitly links these interactions to the critical N inputs that these species provide. Overall, this knowledge gained through this work will directly improve our understanding of the drivers of nitrogen availability in grazing lands of the southern United States, which will improve our ability to promote sustainable land management for cattle producers in the region.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
10%
Developmental
20%
Goals / Objectives
Overarching Goal: I will combine field sampling and a greenhouse study to characterize the specificity of legume-rhizobia interactions for five common pasture legume species, and then use this information to understand mechanisms underlying legume coexistence patterns.Seed Grant Objectives:Objective 1: Determine whether five common species of pasture legumes exist along a gradient of specificity for rhizobial partners through observational field surveysto determine the identities of rhizobial strains nodulating with each legume species.Objective 2: Determine whether legume specificity impacts legume species coexistence and landscape distribution by assessing legume and rhizobia co-occurrence probabilities and spatial cross-correlation.Objective 3: Uncover the mechanisms underlying observed legume distribution patterns by determining the ability of each legume species to amplify abundances of their preferred rhizobial partners in the soil environment and the consequences of this soil conditioning for the growth of heterospecific legumes (Two-Stage Plant-Soil Feedback greenhouse assay).
Project Methods
C.1.A. Study System:I will work across five NC Department of Agriculture (NCDA) Field Stations that span from the state's mountains to coast. From across all sites, I will identify the 5 most common legume species based on discussions with site mangers and site visits by the project team. Although the specific legume species chosen for this research are to be determined, likely candidates commonly seeded into NC pastures include cool-season perennials Alfalfa, White clover, and Red clover, warm-season perennial Chinese lespedeza, and cool-season annuals Crimson cloverand Korean clover.C.1.B. Field Plot Layout: At each field station, 4 10x10 m blocks will be established in a square grid within the site's pasture. Within each block, 42 1x1 m plots will be established in the spatial array depicted in the proposal. This spatial array maximizes the spread of the pairwise distances among all sampling points, while maintaining the minimum number of samples possible to allow for feasibility. GPS locations for all block and plot corners will be recorded to the nearest 10 cm so plots can be revisited in the future.C.1.C. Field Legume and Rhizobial Community Composition: Percent cover of each legume species in each plotwill be estimated to the nearest 1% in June, along with percent cover of other key functional groups. The locations of legume individuals will be mapped to a 10x10 cm grid within each plot and the identities of any legumes present within 1 m of plot edges will be noted. For each legume individual identified at a site, I will calculate the pairwise distance to every other legume individual both within and across plots using each plant's location data (i.e., distances ranging from 10 cm for adjacent legumes to 28.3 m between diagonal corners of the entire sampling layout at the site). These distances will be paired with information on each species' rhizobial specificity to determine the effect of specificity on coexistence.For each of the 5 legume species surveyed, 40 individuals will be selected from plots to encompass individuals growing in isolation (>2 m) from any other legume species and individuals growing together with other legume species in all possible pairwise combinations, limited only by the natural co-occurrence of the legume species in the field. Each selected individual will be unearthed and taken back to the lab, where all root nodules will be excised, grouped by plant individual, surface sterilized, crushed, and frozen at -20 C. DNA will be extracted from the crushate of each plant individual and sequenced at the nifH gene region using high-throughput meta-barcoding on a MiSeq Illumina platform with 300bp paired-end reads. Raw sequences will be demultiplexed and quality reads assigned to amplicon sequence variants (ASVs) using QIIME2. To characterize the composition of the soil-dwelling rhizobial community, three soil samples will be collected to a depth of 10 cm within each plot and composited by plot. DNA will be extracted and sequenced from homogenized soil samples as described above.C.1.D. Classification of legumes along the generalist-specialist mutualism spectrum: For each legume species, their specialization for rhizobial partners will be calculated as the ratio of the realized rhizobial associates identified from each legume species relative to the total pool of rhizobial associates identified across all legume species. These ratios will be bounded between 0 and 1, with lower values indicating more specialist legumes and higher values indicating more generalist legumes.C.1.E. Two-Stage Plant-Soil Feedback Assay: In a greenhouse assay, 10 replicates of all possible pairwise permutations of the 5 legume species (N=25 permutations; N=250 total plants) will be grown to assess plant-soil feedbacks among species that span the generalist-specialist continuum, including specialist-specialist, generalist-generalist, and generalist-specialist pairings, as well as conspecific pairings. Stage 1 - Legume specialization shapes soil rhizobial communities: Field collected soils (Section C.1.C) will be homogenized across all 5 field sites and mixed in a 1:10 ratio with sterile sand to fill sterile Deepots within which legume seeds will be surface sterilized and sown individually (N=50 individuals per species). Plants will be grown for 16 weeks post-inoculation, then harvested to obtain above- and belowground biomass and nodule number. Root nodules will be excised from the root system, pooled by plant, crushed together, and frozen at -20 C for future processing. DNA will be extracted separately from the nodules of each plant individual and a small subsample of homogenized soil from each pot, and sequenced as described above. Soils from each pot will be retained for Stage 2 of the assay, with careful sterile protocols during harvest to prevent contamination across pots. Stage 2 - Soil rhizobial communities impact subsequent legume performances: Legume seeds will again be surface sterilized and planted individually into the pots conditioned in Stage 1 such that all 25 possible permutations of legume species are represented with 10 replicates each (i.e., plants of each legume species will be grown in soil conditioned by plants of each legume species during Stage 1). Plants will be grown for 16 weeks, then harvested to obtain above- and belowground biomass and nodule number, as well as nifH sequencing of nodules and soils, as described above.C.1.F. Covariates: Significant research has investigated environmental tolerances of various legumesand rhizobia. Abiotic drivers of legume and rhizobial diversity include soil moisture, temperature, pH, and N availability. To empirically isolate the effects of rhizobial diversity and specialist/generalist neighbors from the effects of abiotic soil factors, I will measure soil temperature and moisture with a DynaMax soil probe, soil pH using a slurry of deionized water and soil, and soil ammonium and nitrate levels using KCl extractions in every plot, with measurements occurring simultaneously with community composition measurements.C.2. Data Analysis: A vast array of statistical analyses will be possible with the collected data, a subset of which are presented here due to Seed Proposal space limitations. Richness of the nodulating rhizobial community will be compared across the legume species using individual-based rarefaction curves and Chao richness estimates at the species and ASV (97% similarity) levels. The composition of the rhizobial community associated with each legume species will be utilized to determine rhizobial specificity (Section C.1.E). Legume and rhizobial species composition at each site will be separately assessed across plots using PCoA, with measured covariates as environmental factors. Legume community composition will be compared to soil rhizobial community composition using Hierarchical Bayesian Joint Species Distribution Modelingusing the Hmsc package in R, which allows for comparison of species responses to the environmental covariates, as well as the probability of association among legume species and rhizobial strains. Spatial cross-correlation among legume richness, legume abundance, soil rhizobial richness, and measured covariates will be compared using multivariate covariograms. Separate mixed-effects models will be used to assess the consequences of rhizobial specialization for plant growth in Stages 1 and 2 of the Plant-Soil Feedback assay, with plant biomass as a response variable, focal legume specialization status as a fixed effect, and greenhouse position as a random effect. In the Stage 2 model, specialization status of both the first (conditioning) legume and the second (response) legume will also be included as interacting fixed effects.