LONG-TERM PHOSPHORUS CARRYOVER IN LOBLOLLY PINE PLANTATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028185
• Grant No.: 2022-67013-36864
• Project No.: NCZ120464
• Proposal No.: 2021-09941
• Program Code: A1102
• Project Start Date: Feb 1, 2022
• Project End Date: Jan 31, 2026
• Grant Year: 2022

Project Director
Cook, R.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
(N/A)

Non Technical Summary
Loblolly pine (Pinus taeda L.) is one of the most intensively managed and economically important forest species in the world. In the southeastern US, phosphorus (P) inputs at plantation establishment are often required to optimize production, but our understanding of complex interactions among soil chemical properties, the soil biological processes that control nutrient availability, and the influence of previous fertilization rates remains limited. We know pine-associated ectomycorrhizal fungi explore soil volume and enhance nutrient availability to trees but are uncertain how fertilization practices affect the soil-plant-microbial system to alter nutrient availability both in the short and long term. We have the unique opportunity to utilize long-term, replicated field trials in loblolly pine plantations that received increasing rates of P application in the previous rotation and are currently being harvested and replanted. We will experimentally manipulate establishment P fertilization in these geographically-distributed sites with a wide range of soil characteristics to investigate mechanistic underpinnings determining soil nutrient supply. Through a combination of techniques such as microdialysis, ion-exchange membranes, selective dissolution analysis, fractionation of P sub-pools, and characterization of the fungal microbiome and mycorrhizal biomass, we will evaluate the abiotic and biotic drivers of P availability, with loblolly pine growth response as the final bio-indicator. Our goal is to enhance the capacity to predict nutrient limitations and crop tree growth response to fertilization. This work is directly relevantto enhance productivity, soil health, and environmental stewardship.

Animal Health Component

0%

Research Effort Categories

Basic

90%

Applied

5%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0110	1060	50%
123	0611	1070	50%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0110 - Soil; 0611 - Conifer forests of the South;

Field Of Science
1060 - Biology (whole systems); 1070 - Ecology;

Keywords

loblolly pine plantations

microbiome

phosphorus fertilization

soil management

Goals / Objectives
Theoverallgoal of this project isto increase loblolly pine forest productivity and value through understanding and optimizing soil-plant-microbial interactions.The central hypothesisis that the soil supply of nutrients is influenced by chemical and biological characteristics that will interact with previous rotation P fertilization rates in loblolly pine plantations and affect subsequent rotation P availability and pine growth. We will address these issues through a multi-faceted approach that examines novel and traditional techniques to quantify plant-available P across a range of soil characteristics that have been treated with different P fertilization regimes. We plan to utilize data collected via these methods to establish a quantitative basis for a site-specific decision support system to predict the growth response of loblolly pine to P fertilization at establishment. By successfully doing so, this project will increase the precision and economic efficiency of nutrient management decisions across the most significant timber resource in the US. Our objectives in support of this goal involve characterizing both abiotic and biotic contributions to available P and their effects on loblolly pine growth to develop improved fertilizer application guidelines for forest managers.Specifically, we propose to: 1) Evaluate abiotic solid-solution phase dynamics of P as measured by simulated mass flow and diffusion, selective dissolution analysis to characterize reactive secondary minerals, and fractionation of P sub-pools associated with those minerals to determine how fertilizer inputs from previous rotation applications interact with soil characteristics to influence bioavailability,2) Characterize how the fungal microbiome and mycorrhizal biomass are affected by previous rotation and current rotation fertilization to identify specific fungal taxa known to accumulate and exchange P with loblolly pine, and3) Evaluate abiotic and biotic influences on nutrient acquisition and growth response of loblolly pine in the field and under controlled laboratory conditions.We will leverage a network of field sites in the southeastern USwith a range of intensities of sampling. The deliverable to forest industry stakeholders based on the results will be a decision support tool that guides land managers on appropriate testing methods and guidelines for establishment P fertilizer application based on previous fertilization practices and soil characteristics determined in this study.

Project Methods
The Efforts that will create a change in knowledge of P fertilization requirements and the action of applying fertilization at establishment in loblolly pine in forest industry include the following methods. We will Evaluate the impact on the industry based on surveyed fertilizer application changes and modified practices by forest managers.Proposed Activities and Techniques: Objective 1 Abiotic driversIon-exchange membrane probes(field activity) assess deficiencies and surpluses of P and show correlations with bioavailable P. Due to the relatively low nutrient concentrations in our soils, these probes will be cycled every four months in the field and compared to traditional Mehlich-III and novel microdialysis extractions. Four sets of probes will be placed in each of the plots for each of the three intensive sites and analyzed with high-performance liquid chromatography.Microdialysis(lab and field activity) simulates diffusion and mass flow of plant-available nutrients from the rhizosphere to the plant root and can be used non-destructivelyin situor with soil samples collected from the field. At extensive sites, we will collect intact soil cores at site establishment from 0-15 and 15-30 cm for each treatment to be analyzed in the lab. At intensive sites, samples will be collected from 0-15 cm each year andin situmicrodialysis extractions will occur in the 0-15 cm depth in undisturbed soil. Extracts will be analyzed with ultra-performance liquid chromatography.Chemical Extraction(lab activity) using traditional Mehlich III will be the standard of comparison to the novel techniques. Mehlich-III extracts (Mehlich, 1984) will be analyzed by ICP-OES. Composite soil samples will analyzed from 0-15 and 15-30 cm in each plot. Additionally, we will characterize soil mineralogy and the potential bioavailability of P using universal laboratory dissolution and fractionation techniques on samples taken at planting on both extensive and intensive sites. As mineralogical proxies, selective dissolutions (ammonium oxalate, dithionite-citrate, and sodium pyrophosphate) of Fe, Al, and Si will be performed following standard USDA NRCS protocols.Expected Results: We expect that microdialysis and ion exchange membrane extractions will provide a more complete picture of plant-available P than traditional Mehlich-III extractions by simulating mass flow and diffusion P, and will correlate with modified Hedley fractionations for P and with mineralogical proxies. Better predictions of nutrient availability will allow landowners to optimize fertilization practices to promote sustainable site management.Data analysis:Data will be analyzed using generalized linear mixed models to develop relationships between sampling and analysis methods, nutrient availability, and fertilization regimes.Objective 2: Biotic DriversFungal bags(field activity) consist of 52 µm nylon mesh bags filled with acid-washed sand. The mesh size is fine enough to allow the fungal mycelium to enter but omits plant roots. A total of 16 bags, eight control sand-only bags, and eight "baited" bags, containing sand plus P in the form of apatite, will be placed in each plot at each site for 6-month intervals.Fungal biomass and community structure will be sampled from mesh bags. Hyphae will be homogenized, and a subsample will be taken for each treatment for DNA extraction. Two methods will quantify abundance: Quantitative PCR (qPCR) analysis and Loss on Ignition. To determine which fungal communities are present in the bag vs. the rhizosphere, next-generation sequencing will be used to characterize the fungal microbiome, both for diversity and taxonomic structure.Expected Results: We expect differences in fungal biomass due to P availability. We expect there to be a critical level at which the ECM fungal community structure will shift in relation to nutrient availability and that fungal bags could serve as an indicator of site-specific P limitations.Data analysis: For fungal biomass samples, treatment effects will be analyzed for a non-linear response or analyzed with a generalized linear mixed model. Non-normal samples will be analyzed via Kruskal-Wallis nonparametric tests.ECM fungal community data will be analyzed using raw ITS amplicon sequences processed using phyloseq. De-noising and de-replicating sequences will generate tables of amplicon sequence variants (ASV) and will be performed using DADA2 to provide more detail than operational taxonomic units. After this processing, fungal ASVs will be taxonomically classified using the UNITE reference database. Pairwise similarity matrices for the microbiome structure will be generated from Euclidean and Bray-Curtis distances and visualized using NMDS. Differences between treatment plot rhizosphere soil and mesh bags will be tested via PERMANOVA.Objective 3: Growth ResponsePlant growth responsewill be evaluated with tree measurements conducted annually in winter. Root collar diameter will be measured until the diameter at breast height can be measured. Total height, height to base of the live crown, and damage and mortality assessments will be made on all measurement trees. Foliar nutrient concentrations will be determined annually during the dormant season from samples collected from five co-dominant, healthy trees in each measurement plot and combined for plot-level analysis.Plant nutrient acquisition will be tested onsoils collected from each of the treatment plots and brought back to the lab to determine the relationship between the native ECM fungal community and the ability of loblolly pine to acquire nutrients through their symbiotic hyphal networks. Custom-made two-compartment boxes, with a mesh measuring 52 µm to exclude plant roots, will be used to separate the seedling (left compartment) from the fertilizer source (right compartment), allowing only fungal hyphae to pass through. Fertilizer treatments mirroring our field treatments will be added to the side of the box where only ECM fungal hyphae can enter, allowing for the detection of different nutrient acquisition capabilities ECM fungal communities from different field treatments. Loblolly pine seedlings will be destructively sampled two months later, enough time for functional ECM symbiosis to occur, to determine total nutrient acquisition.Both foliage samples and destructively harvested loblolly pine seedlings will be analyzed for nutrient concentration.Expected Results:We expect that field plots receiving less P fertilization in the previous rotation, and that did not receive P at re-planting (Low-wo/P), will show less growth over time than plots that receive greater P in the previous rotation. We expect the time for those growth curves to diverge will be dependent on site-soil characteristics. Novel methods of abiotic and biotic nutrient availability will explain the magnitude and duration of loblolly pine growth response with more accuracy than traditional methods alone. Soil properties at each site, as detailed by our selective dissolution and fractionation analysis, will influence both the magnitude and duration of pine growth response, providing better information to predict nutrient limitations and response to fertilization in the future. Split-screen fungal boxes will show how shifts in fungal communities affect the acquisition of P in a much shorter time period.Data Analysis:Lasso regression will be used for variable selection to create simplified models to explain how abiotic and biotic variables predict growth response and nutrient acquisition in loblolly pine. Mixed models will be used to analyze growth response of trees over time in the field with regard to treatment effect of providing P fertilization at establishment.

Progress 02/01/23 to 01/31/24

Outputs
Target Audience:The primary target audience includes forest industry and nonindustrial private landowners. We delivered science-based information derived from this research in the following activities: 1) Jacob Hackman, PhD now graduated fromthe project, delivered a 20 minpresentation to forest industry (75 in attendance) a the Forest Productivity Cooperative Annual Meeting in Blacksburg NC (Aug2023); through his PhD defense (~30 in attendance including forest industry in May 2023); and at a Fall contact meeting in Jekyll Island, GA (~20 in attendence, Nov 2023, 3) Presentation of preliminary results directly to forest industry through workshops including for continuing education credits. Dr. Cook presented to RMS in Wilmington, NC in Dec 2023 (1 hour presentation, 4 in attentendence), BTC in Colombia, SC (1 hour field presentation at a P carryover site, 12 in attendence), Camcore at Hofmann Forest NC in Oct 2023 (~30 in attendence), AFM at Panama City Beach, FL (~35 in attendence, 1 hour presentation), JD Irving in Bangor ME in Sept 2023 (~10 in attendence),Molpus Timberland Management in Valdosta, GA in Dec 2023(1 hour field presentation, 22 in attendance). Changes/Problems:It took some time to recruit and start the new phd students, so a NCE may be required. We will also be asking for a rebudget as we have already spent almost all the supplies out of the NCSU budget and may need to reallocation from other lines. What opportunities for training and professional development has the project provided?Jacob Hackman, PhD student now graduated from the project, has published two papers from his PhD concerning the evaluation of soil tests and their correlation with growth response. Dan Hong and David Enemo, PhD students at VT are actively developing their protocols to assess the relationship between forest floor quality and quantity and P availability, and P dissolution from soils across the experiment. 2) Jacob Hackman has submitted revisions to his PhD paper regarding the influence of P treatment at two sites on the fungal microbiome. Jacob Hackman isnow workingas a post doc in our lab and interviewing with potential employers. He interviewed for an academic position at University of Georgia and currently has an interview for University of Arkansas. How have the results been disseminated to communities of interest?Yes, results have been disseminated through several presentations, the FPC Annual Meeting in Aug 2023, the Fall Contact meeting in Nov 2023, and many field visits with forest industry in the fall of 2023 (Molpus, AFM, RMS, BTG, Arborgen, Jordan Lumber, JD Irving, Camcore). Forest industry stakeholders were also present online for the dissertation defense of Jacob Hackman's work that is detailed above. Poster presentations of the results include: David C. Enemo, Daniel S. Hong, David Carter, Rachel Cook, and Brian D. Strahm. 18-year Effects of Nitrogen and Phosphorus Fertilization on Selected Soil Chemical Properties under Loblolly Pine Plantations in the Southeastern United States. 14th North American Forest Soils Conference. Eugene, OR. July 16-20, 2023. Daniel S. Hong, David C. Enemo, Rachel L. Cook, David R. Carter and Brian D. Strahm. Loblolly pine responses to carryover phosphorus fertilization in the southeastern US. 14th North American Forest Soils Conference. Eugene, OR. July 16-20, 2023. David C. Enemo, Daniel S. Hong, David Carter, Rachel Cook, and Brian D. Strahm. 18-year Effects of Nitrogen and Phosphorus Fertilization on Selected Soil Chemical Properties under Loblolly Pine Plantations in the Southeastern United States. Forest Productivity Cooperative Annual Meeting. Blacksburg, VA. August 16-17, 2023. What do you plan to do during the next reporting period to accomplish the goals?We have four phd students (two students funded with leveraged support) actively working on each of the objectives for the project. After the first three publications, we have another one in prep that will be submitted this year. We are about to install two more field trials and are quickly accumulating data from our network of trials across the southeast.

Impacts
What was accomplished under these goals? The issue this project addresses is the ability to predict under what circumstances fertilizer P applied in one rotation of loblolly pine will remain available in the following rotation. Results will help forest managers and landowners to make better management decisions in loblolly pine plantations by either not applying P fertilizer when it is not necessary or by recommending P fertilizer when it is required, improving both environmental and economic outcomes. Regarding each specific objective: 1)Microdialysis, a new method taken from neuroscience, uses tiny probes to simulate the process of mass flow and diffusion of nutrients in soils. These are the primary ways that plants take up nutrients. We used microdialysis on soils collected from plots treated in the field with phosphorus at varying levels in the last rotation (0, 40, 60, and 121 kg/ha P), and a subset re-fertilized at establishment (40 kg/ha P in the previous rotation and 45 kg/ha P in the current rotation), to assess the availability of phosphorus to the trees. We correlated the "available" P, as measured by microdialysis to tree growth to look for relationships. In the clay soil, we found the highest concentrations in the highest fertilizer treatment one year after establishment but no differences among the other treatments. In the sandy soil, the greatest difference from year 0 to year 1 was also in the highest (121 kg/ha P) treatment. We also inserted these probes into the tree itself to evaluate if we could see actual nutrient uptake in real-time within the tree and if this was affected by different levels of residual nutrients from fertilizer applications. Due to rates of evapotranspiration, sunny days pulled much more water through the tree, but when we accounted for those differences, we saw weak evidence (p = 0.23) that P fertilizer treatment affected P concentration in the tree, but indicated that future work to investigate this further would be worthwhile. Great control of when data is collected based on weather would likely help establish a stronger relationship. A paper has recently been published further detailing these results (Hackman et al., 2023 in Plant Soil). 2)We buried "fungal capture bags," collected samples from tree roots (the rhizosphere), and samples from bulk soil to assess methods of collecting fungal communities with two of the first sites to be installed (a poorly drained clay soil - an Alfisol, and a somewhat poorly drained sandy soil - a Spodosol). Because of the intensity and cost of running fungal microbiome analysis, we selected a subset of plots (No fertilizer, 40 kg/ha P in the previous rotation, 121 kg/ha P in the previous rotation, and 40 kg/ha P in the previous rotation plus 45 kg/ha P in the current rotation) to assess the effect of carryover and newly applied P fertilizer on the fungal community. First, we found in the mesh bags, that bags "baited" with P rather than just pure sand, had greater amounts of fungal biomass inside. These bags have a very fine mesh that allows fungi to enter but not tree roots. So we determined that the fungi do seek out the P in the bags. We buried these bags before the previous rotation was harvested (in 2018) and through new plantation establishment up to 2022. Interestingly, there was more difference in fungal biomass among treatments (with the control having more fungal biomass in bags) in the mature plantation before harvest than in later samplings. We suspect this may be because the ECM fungal population (those that are symbiotic with loblolly pine) likely declines rapidly once the trees are harvested (since they depend on the trees for sugar). It will be interesting to see in future mesh bag sampling if the differences across P fertilizer treatments return with time. We also looked at the diversity of the fungal community among treatments at the two sites. Contrary to what many might expect, the treatment that received fertilizer had greater fungal diversity than the control, but only at the clay site. At the sandy soil site, there were no differences due to P fertilizer treatment in diversity. So we can conclude, so far, that P fertilization does not negatively affect fungal diversity. Finally, we looked at the relative abundances of all fungal species and found that site (clay vs sandy soil) was a much bigger driver than P fertilizer treatment. Although when we looked just at the ECM fungi, there was not as much difference between sites. These results have been submitted in a publication to the journal Mycorrhizae and is currently under review. Next, Ben Rose, the new PhD student on the project is systematically sampling all the field sites to characterize the differences across all sites and treatments. Four sites have currently been sampled and the rest will be sampled this winter. This analysis will expand the original analysis conducted on the first two sites. Additionally, another new PhD student, Maria Higuita, is assessing the impact of synthetic fungal communities under lab conditions on P uptake by loblolly pine. The question is to see if loblolly pine grows better with single or multiple fungi present, i.e. do the fungi each play a slightly different role. Preliminary data with one vs two fungi suggest that two fungi may have a synergistic effect on nutrient uptake. 3)In field conditions at the first two sites established on contrasting soils (a clay soil and a sandy spodic soil), at year 2 we already are seeing significant differences in growth due to treatments (amount of P applied in the previous rotation and some plots receiving additional fertilization at planting). So far the Mehlich 3 soil test helped to predict height growth in the clay soil but not in the sandy soil. Interestingly, the O-horizon (also known as the forest floor) from the previous rotation also appears to be a good predictor of age 2 height of trees. Plots that received more fertilizer in the previous rotation also tended to have greater O horizon, meaning that the forest floor appears to be a reserve of nutrients released to the next rotation. Interestingly, so far year 3 measurements suggest that as long as at least 50 lbs of P were applied (either in the previous rotation or as fertilizer in the current rotation). These results have been accepted for publication in the journal Forest Ecology and Management. In our microdialysis experiments in lab conditions, we tested soils from the first two field sites to be established in a new plantation and found that in the clay soil there was a positive relationship between diffusive (microdialysis) P concentrations and tree heights at age 2 but no relationship in the sandy soil. There was also much more diffusive P in soil solution in the sandy soil, likely because there are fewer sites for it to "attach." Furthermore, because these samples were taken from the surface soil (top 15 cm), we decided to sample soils from deeper in the soil profile in case more P was concentrated at greater depths. Those samples have just finished being collected in the lab and are now being processed for diffusive P concentration. These two soils represent two very common soils planted in the southeast but there are many more sites to analyze with different soils. More samples are being taken with every new site installation. Five sites have been installed (meaning the former rotation has been harvested and new trees have been planted) and trees are getting measured every winter. Three more sites are still to be installed. Additionally, Jacob Hackman is now working as a postdoc and using O horizon and soil data collected previously in 2013 down to one meter to assess the abiotic influences (texture, Al Fe oxides, etc) on the build up of P in the soil horizons and any relationships with current tree growth.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Hackman, J., R.L. Cook, K. Garcia, D. Carter, A. Woodley, B. Strahm, T.J. Albaugh, R. Rubilar, O. Campoe. Pinus taeda Carryover Phosphorus Availability on the Lower Atlantic Coastal Plain. Forest Ecology and Management. In press.
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Hackman, J., R.L. Cook, B. Strahm, D. Carter, A. Woodley, and K. Garcia. Using microdialysis to assess soil diffusive P and translocated sap flow P concentrations in Southern Pinus taeda plantations. Plant and Soil. Published online 05 Jan 2024
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Hackman, J., R.L. Cook, K. Garcia, A. Woodley, D. Carter, B. Strahm, C. Averill, R. Vilgalys. Fungal Biomass and Ectomycorrhizal Community Assessment of Phosphorus Responsive Pinus taeda Plantations. Mycorrhiza. Under review.

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:The primary target audience includes forest industry and nonindustrial private landowners. We delivered science-based information derived from this research in the following activities: 1) the PD provided a lecture on P carryover and explanation of the experimental design in FOR 713 Advanced Silviculture to 7 graduate students, 2) Jacob Hackman, PhD candidate on the project, delivered a 20 min presentation to forest industry (75 in attendance) a the Forest Productivity Cooperative Annual Meeting in Raleigh NC (Aug 10, 2022),3) Presentation of preliminary results directly to forest industry through workshops including for continuing education credits. Dr. Cook presentedto Molpus Timberland Management, Lufkin, TX Oct 26, 2022 (1 hour field presentation, 22 in attendance), Potlatch Deltic in Prattville, Alabama Dec 8, 2022 (10in attendance), Campbell Globel in Wilmington, NC Nov 3, 2022 (9in attendance), and Rayonier in Kinder, LA Nov 17, 2022 (5 in attendance). Changes/Problems:It took almost a year to recruit, identify and start two new PhD students at Virginia tech. Both PhD have started as of Jan 2023 (one year after grant start date). This will likely contribute to a need for a no-cost extension in the future. At NC State, a former graduate student, involved in all the preliminary work and grant writing should graduate Spring 2023 and a new PhD with excellent fungal microbiome skills has agreed to start in the fall. What opportunities for training and professional development has the project provided?The PhD student on the project, Jacob Hackman, had the opportunity to work directly with Rayonier, one of the forest industry partners that hosts two field sites and learn about industry research opportunities. Additionally, the field skills he is developing and the work he has been performing helped land him an interview at another large university for a potential assistant professor position where he was able to present his work on P carryover and develop his professional interviewing skills. How have the results been disseminated to communities of interest?Yes, results were disseminated through Jacob Hackman's presentation at the Forest Productivity Cooperative annual meeting and are currently in near final draft form for a paper submission. What do you plan to do during the next reporting period to accomplish the goals?The manuscripts Jacob Hackman is preparing to finalize his dissertation include 1) comparison of soil mehlich 3 extractions, resin probe extractions, and tree growth, 2) microdialysis P availability, and 3) fungal microbiome from the first two sites that have been installed, planted, and most thoroughly measured. We will also bring on a new PhD student in Fall 2023 to replace Jacob as the primary graduate student at NCSU.

Impacts
What was accomplished under these goals? Regarding each specific objective: 1) Jacob Hackman (phd student) has just finished the microdialysis extractions to asses solid-solution phase P dynamics due to carryover vs recently fertilized P and samples are currently being analyzed. Resin Probe P from two sites (FL, GA, and NC) are complete and results are currently in draft form for a manuscript. Additionally, another siteis being installed this winter. 2) Fungal biomass has been shown through fungal bags to preferentially target bags baited with phosphorus and we are finding some differences in biomass among treatments. Data are currently under review and more fungal bags are being prepared to be deployed. Microbiome results have just arrived and are currently under analysis for the first two sites. 3) Measurements at the three intensive sites (NC, GA, and FL) have been taken for this winter and data will be analyzed with soil biotic and abiotic factors. Importantly, two new PhD students (one funded by VT along with the USDA graduate student) have started this fall and will begin on the P fractionation portion of the experiment and help with site installation and data collection. The former PhD working on the project should graduate this spring at NC State, and a new PhD student has been identified to replace him.

Publications