Performing Department
(N/A)
Non Technical Summary
The longleaf pine (LLP) savanna ecosystem has great potential to serve as a multifunctional landscape, contributing to both economic and environmental sustainability in the southeastern US. Yet <4% of this once expansive ecosystem remains; thus, methods for restoration and management of LLP savanna are critical. Extant LLP savannas are nutrient poor, limiting productivity of both the LLP timber trees as well as the understory preventing successful restoration of the plant-fire feedback loop. With this SEED GRANT, we aim to begin to understand the role of two key microbial symbioses in promoting nutrient availability and ultimately improving functioning of the LLP ecosystem: (1) rhizobial bacteria critical for nitrogen fixation in legumes (plants in Fabaceae) and (2) mycorrhizal fungi critical for phosphorous uptake in many plant lineages. Our research will advance current priorities of BNRE Sustainable Agroecosystems (A1451) by focusing on improving productivity in managed LLP savanna. This SEED GRANT will support (1) observational data collection, (2) establishment of an experimental platform manipulating the frequency of nutrient-based mutualisms, and (3) the formation of a network of sites for future LLP research. This work will lead to increased understanding of nutrient-based mutualisms with the long-term goals of ensuring adequate fuel for fire spread and enhancing understory and tree production, and will form the basis for a future proposal for a Standard USDA grant. Overall, we aim to yield innovative and environmentally-sound management strategies for the LLP ecosystem, thereby improving the environmental health and sustainability of our natural resource base.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Overarching Goal: Assess the potential of nutrient-based mutualisms to promote nutrient availability, biomass production, and restoration success in the longleaf pinesavanna ecosystem, with the ultimate long-term goal of promoting sustainable management practices in this economically and environmentally important ecosystem.SEED GRANT Objectives(1) Observationally assess the frequency and identity of understory plant-microbial mutualisms (i.e., legume-rhizobia and plant-mycorrhizae) in a nutrient poor longleaf pinesavanna;(2) Experimentally assess impacts of nutrient-based mutualisms on longleaf pineunderstory function by manipulating the abundances of legume seeds, rhizobial bacteria, and mycorrhizal fungi;(3) Visit five additional longleaf pinesites along the latitudinal gradient of the longleaf pineecosystem to collect baseline mutualism frequency data and develop a network of sites for future research.Overall, the proposed research will increase understanding of the role of nutrient-based microbial mutualisms in promoting health and maintenance of longleaf pinesavanna. Building off the experimental platform and network of sites developed through this SEED GRANT, as well as preliminary patterns identified from the data we collect, we will develop a proposal for a Standard USDA grant to expand our understanding of how altering the abundances of nutrient-based mutualisms influences soil nutrient concentrations, understory dynamics, and ecosystem productivity (including herbaceous layer and longleaf pinetree seedling growth). Increasing our understanding of these interactions is critically important for maintaining the economic viability of longleaf pineproduction in the US, while utilizing our natural resources in a sustainable way.
Project Methods
Focal Study Site: The majority of the work proposed here (Obj. 1 & 2) will occur within the Sandhills ecoregion of North Carolina on a portion of the 60,000-acre Sandhills Game Land.Objective 1: Observationally assess the frequency and identity of understory plant-microbial mutualisms (i.e., legume-rhizobia, plant-mycorrhizae) in a nutrient poor LLP savanna (Year 1).Identify Dominant Plant Species: At our focal LLP site, we will establish 72 3x3 m plots arranged in a grid with 3 m aisles between plots, within which we will measure percent cover of each plant species within a permanently marked 1x1 m quadrat using a modified Daubenmire methodin June of Year 1. From this data, we will determine the Dominance Candidate Index (DCi), a metric that incorporates both species frequency across plots and average cover within plots. The 5 grass, 5 leguminous forb, and 5 non-leguminous forb species with the highest DCi values will be determined and thereafter considered dominant species within each functional type for the purposes of this work (N=15 species across all functional groups).Plant Excavation & Soil Collection: We will then observationally determine rates of mutualistic interaction with mycorrhizae (all dominant species) and rhizobia (legumes only). For each species, 10 individuals will be randomly selected from outside of the established plots and carefully excavated, keeping root systems intact (N=150 individuals across all species). For half of the legume individuals of each species (5 individuals per species, 25 individuals overall), a subsample of up to 10 randomly selected nodules from each individual will be immediately removed for Acetylene Reduction Assays to estimate of nitrogenase activity and therefore a proxy for N-fixation. The remainder of the plant roots and the intact root systems of all other individuals sampled (half of the legume individuals and all of the non-leguminous species) will be wrapped in damp paper towels and stored in individually labeled zip-sealed bags for transport on ice back to the lab.The sampled nodules will also be transported back to the lab for sequencing to identify the rhizobial strains contained within.Lab Processing: All collected roots will be carefully washed under low-pressure running water in the lab. For legumes, the total number of nodules per individual will be counted as a measure of frequency of rhizobial associations. Additionally, five 5 cm subsections of roots from all individuals of all species will be stained with Typan-Blue and scored for root colonizationto assess frequency of mycorrhizal associations per individual. Within one week of collection, the legume root nodules for each plant individual used in the ARAs will be surface sterilized and crushed in 100 μl sterile water. Additionally, whole root samples (not including nodules) will be subsampled (5 g) for all plant individuals, surface sterilized, and ground to a fine powder using liquid nitrogen. DNA will be extracted from nodule crushate (targeting rhizobial associations) and whole root samples (targeting mycorrhizal associations) using Qiagen DNeasy PowerSoil HTP 96 kits.Objective 2: Experimentally assess impacts of nutrient-based mutualisms on LLP understory function by manipulating the abundances of legume seeds, rhizobial bacteria, and mycorrhizal fungi (Years 1-2).Establish Experimental Treatments: Following the observational (pre-treatment) collection described in Obj. 1, we will apply six experimental treatments within the 72 previously established 3x3 m plots in a randomized block design (n=12 per treatment). All treatments will be applied in June of Years 1 and 2. (1) Unmanipulated Control. (2) Legume Seed Addition.(3) Rhizobial Bacteria Addition.(4) Mycorrhizal Fungi Addition.(5) Combined Legume Seed, Rhizobial Bacteria, & Mycorrhizal Fungi Addition.(6) N & P Addition.Frequency & Function of Nutrient-Based Mutualisms: Individuals of the 15 dominant species will be excavated from five randomly selected experimental plots of each treatment at peak biomass, with care taken to minimize disturbance to the remaining plot area. These individuals will be assessed for nodule number and fixation rate (for legumes), mycorrhizal root colonization rates (for all species), and leaf nutrient content. Resin bags will be deployed within each plot April-October in Year 2 to assess integrated soil N and P availability.Objective 3: Visit five additional publicly and privately owned LLP sites along the latitudinal gradient of the LLP ecosystem to develop a network of sites for future research (Year 2). This SEED GRANT will lay the foundation for a future proposal addressing nutrient-based mutualisms in LLP savanna along its latitudinal range. In Year 2, the PDs and postdoctoral scholar will visit the five additional LLP sites that span this range, including one site in North Carolina (Bladen Lakes State Forest), South Carolina (Santee Experimental Forest), and Georgia (Fort Stewart), and two in Florida (Olustee Experimental Forest, Ocala National Forest). We will use the visit to network with on-site managers and researchers, share our findings from Objs. 1 and 2, and informally survey on-site contacts for their view of mutualisms in LLP savanna management, gaining critical shareholder input for the future proposal. We will additionally collect preliminary observational data from each site on (1) plant community composition from 20 m2 plots randomly spread throughout the site, (2) soil N and P levels within each plot, and (3) collect 5 individuals of each of the dominant 5 grass, 5 legume, and 5 non-leguminous forb species from locations across the site to assess mycorrhizal colonization and nodulation (for legumes) rates using the methods described above.Data AnalysisObjective 1: Our initial identification of the rhizobial and mycorrhizal mutualists that associate with 15 dominant plant species will contribute greatly to our understanding of these partnerships. For each plant species, we will calculate the frequency of these mutualisms as well as community-level metrics for the rhizobial and mycorrhizal associates using the R package codyn, comparing across plant species using ANOVAs and PERMANOVA as appropriate.Objective 2: Treatment effects on univariate response metrics (e.g., understory biomass, soil N and P, plant richness) will be assessed using ANOVAs. Long-term, the experimental platform will be used to link the effects of nutrient-based symbioses on LLP function through Structural Equation Modeling (SEM) based on a priori null models of the interactive effects of components of the plant and microbial communities on measures of plant tissue chemistry, soil health, and primary productivity (future Standard Grant proposal).