DRIVERS AND INDICATORS OF SOIL HEALTH IN SEMI-ARID AGROECOSYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1015239
• Grant No.: 2018-67019-27799
• Cumulative Award Amt.: $498,000.00
• Proposal No.: 2017-05335
• Project Start Date: Mar 1, 2018
• Project End Date: Feb 28, 2023
• Grant Year: 2018
• Program Code: [A1401]- Foundational Program: Soil Health

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Soil and Crop Sciences

Non Technical Summary
Our ability to feed a growing human population depends on healthy soils that can sustain crop production in the face of water scarcity and increasing environmental stress. Soil health is realized when soil carbon (C) is accrued and stabilized, while nitrogen (N) is tightly recycled to sustain crop production. We hypothesize that this can be realized in dry-land agroecosystems by continue and diverse cropping systems, which include a legume in the rotation. This is because continue inputs support soil C accrual and soil organic matter aggregation, while the incorporation on N-rich labile inputs via the legume would increase N availability and microbial efficiency, and the two combined promote beneficial microbial network connectivity, overall resulting in increased soil C stabilization and N recycling, thus regenerating soil health. We propose to test our overarching hypothesis through a combination of on-farm monitoring of microbial network and soil structure, field experimental manipulations using 13C and 15N labeled crop residues, and laboratory incubations, which leverage long-term agricultural experiments, with wheat-fallow and continuous crops with and without legumes in the rotation in the semiarid Central Great Plains.

Animal Health Component

50%

Research Effort Categories

Basic

40%

Applied

50%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0110	1070	100%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil;

Field Of Science
1070 - Ecology;

Keywords

n recycling

soil healt

aggregates

crop rotations

microbial network

semi-arid cropping

Goals / Objectives
The long term goals of this project are to: 1) improve our fundamental understanding of the role of the soil microbiome and physical structure as drivers and indicators of soil health in semi-arid agroecosystems, and 2) use this understanding to propose cropping system management strategies that can sequester C while sustaining N recycling, thus regenerating soil health.We have the three following specific objectives:Objective 1: Assess the role of cropping system intensity versus diversity to regenerate soil structure and microbial network connectivityObjective 2: Understand and quantify the ability of semi-arid cropping system intensity versus diversity to solve the soil carbon dilemma?Objective 3: Understand and quantify the role of soil structure (i.e. aggregation) in residue-derived C and N processing and stabilization, and its interaction with the soil microbiome.We will take advantage of the emerging rotations in the Central Great Plains and long-term cropping systems study sites to address our specific research objectives.

Project Methods
Analyses of soil microbial communities, aggregate size distribution, soil C and N stocks and their distribution between POM and MAOM, in cropping systems of increasing intensity and diversity. During the first year of the project we will visit all our twelve field sites and sample soils for the analyses of the microbial community and network structure, aggregate size fractions, total soil C and N stocks, and their distribution among functionally meaningful primary SOM pools, namely a particulate organic matter (POM) and a mineral-associated organic matter (MAOM) pool, using the methods described in the section below. Each of the twelve field sites will be sampled by coring 0-10 cm soil depth at multiple locations within 4 replicate areas (10*10 m) per rotation treatment, making use of established replicate plots where they exist or identifying the 4 replicate areas within fields. Bulk density will also be measured at the time of sampling, by conventional analyses. Soil samples will be composited for each replicate area to obtain a total of 48 soil samples (i.e., 3 rotation managements * 4 fields * 4 replicates). Soil sampling will be coordinated so that it will always take place at wheat heading for each crop rotation, to assure comparability. Field moist soil will be sieved to 8mm, split into subsamples, and stored either air dry for SOM fractionation and elemental analyses, or at -20°C for microbial community structure and network analyses.Production of 13C and 15N labeled wheat residues.We will produce dual labeled wheat residues to use for field and laboratory incubations. Winter wheat seeds will be germinated, vernalized, and grown in our dual isotopic labeling chamber. 13C enriched CO2 is pumped into the sealed chamber constantly to achieve a CO2 concentration of 360-400ppm and a target 13C labeling of 4 atom %. The plants are watered with 15N-labeled Hoagland's solution weekly. Fertilizer amounts are increased as wheat biomass increases, with a target residue C:N ratio comparable to field grown wheat in the region, and a 15N labeling of 7 atom %. The chamber hosts 40 nine-gallon pots, each with its own drip irrigation system, and 4 wheat plants will be grown per pot within a fully inert pot media (i.e., vermiculite, sand and synthetic clay), to avoid isotopic C contamination from the decomposition of SOM. Wheat will be grown to maturity, before the chamber will be opened and the crop harvested. Wheat shoots and root residues will be cleaned, and air dried prior to being used for incubations. Three subsamples for each of the root and shoot residues will be oven dried (70°C) for air dry weight correction, before being pulverized and analyzed for their C and N elemental and isotopic composition in an ElementalAnalyser-Isotope Ratio Mass Spectrometer (EA-IRMS)Analyses of the effects of cropping system intensity and diversity on microbial decomposition of crop residue, N recycling and the formation of residue-derived persistent SOM.We will conduct a two-year field incubation of the dual labeled (13C and 15N) wheat residue in situ in replicated long term plots of each of three cropping systems (WF, CON-L and CON+L) at the USDA-ARS Central Great Plains Experimental station in Akron, CO. The experiment will be conducted using a consolidated approach we have successfully used in several previous experiments. In this study we will use root residues and will incubate them mixed within the 0-10 cm soil layer, free of bags inside PVC collars, at a rate representing average root residue input for the site. Residue will be left in the field to decompose for two years. Three sets of collars will be established, each in 4 replicates, to allow for three harvests, after 3, 12 and 24 months of decomposition. At each harvest, collars will be excavated, and the full soil within collected. All soils will be sieved to 8mm and a subsample from each analyzed for gravimetric water content by mass loss after drying at 105ºC. The root samples remaining on the sieve will be rinsed of all residual soil and oven dried at 70ºC for the estimate of root residue decomposition. A fresh 8 mm sieved subsample of each soil will be used for SOM fractionation, to obtain the aggregate size distribution, and primary SOM fractions (i.e., POM and MAOM) distribution. Additionally, a subsample of each of the 2 mm sieved soil will be stored at -20°C for phospholipid fatty acid (PLFA) analyses. Oven-dry soil and root samples will be pulverized and analyzed for %C, %N, δ13C and δ15N on a EA-IRMS, while PLFA extracts will be analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-co-IRMS). Additionally, at each harvest, we will collect crop roots growing immediately outside all of the collars. Roots will be dried in oven (70°C), pulverized and analyzed for δ15N on an EA-IRMS to compare rotation management effects on crop N uptake from residue N recycling.Laboratory experiments with 13C and 15N residue addition to study the role of soil structure (i.e. aggregation) in residue-derived C and N processing and stabilization, and their interaction with the soil microbiomeWe will establish a laboratory incubation using three soils with high aggregation and contrasting microbiomes. After we complete all the analyses of the soils collected at the 12 field sites, we will select the three soils with the highest mean weight diameter (i.e. most aggregated), and contrasting microbiome and will incubate them with and without dual labeled 13C and 15N crop residue, at an addition rate comparable with crop residue inputs in the field. To optimize labeled residue use, we will use the aboveground wheat residues. In order to asses the effect of aggregation on residue processing we will impose two aggregation treatments to the three soils: Aggregated (8 mm sieved), and non-aggegrated (fully dispersed). Dispersion will be conducted in water to minimize impacts on the microbial community, while ensuring breaking of all macroagrgegates and most of microaggregates. Four replicates of each treatment will be established for a total of 48 experimental units (e.g., mason jars). Two full sets of experimental units (i.e., n=96) will be incubated at optimal temperature (25°C) and moisture (60% water filled pore space) in a constant temperature room, to allow for two destructive harvests, after 3 and 12 months from incubation. At each harvests soils will be analyzed for residue C and N loss, and residue C and N incorporation in total SOM, and as distributed in POM and MAOM fractions. Similarly, we will determine residue-derived incorporation in microbial markers (i.e., PLFA). Additionally at each harvest we will assess the soil microbiome structure for the aggregated and disaggregated soils, to understand and quantify the interactions between microbiome and aggregation on residue decomposition and C and N processing and stabilization.

Progress 03/01/18 to 02/28/23

Outputs
Target Audience:Our target audience are peer scientists, company interested in regenerative agricultural and producers. Results from this project have been widely communicated through publications, presentations at conferences and extensioneventsand have also been incorporated into seminars and lectures for graduate and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity to train and develop professionally one MSc student - Rebecca Even, currently the associate lab manager in theSoil Innovation laboratory at Colorado State University, and a PhD student, Dr. van der Pol, nowthe lead soil scientist at The Land Institute. We alsoemployed and trained in laboratory and field methods a high number of undergraduate students. How have the results been disseminated to communities of interest?Over the course of the project, results were disseminated through several presentations at conferences, invited seminars, field days and other extension events (only PI Cotrufo has given more than 3-4 seminars/webinars a year in the past 4 years), the most relevant of which are reported in the products.We have also presented results for this work internally at CSU at a departmental seminar series. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: To quantify the effect of diversification by legume incorporation and intensification of grain production via perennialization on soil regeneration, we performed a 2-year mechanistic field study in Kernza cropping systems. The intermediate wheatgrass domesticated for grain production as Kernza® (Thinopyrum intermedium) is the first perennial grain available to U.S. growers. We quantified the ability of the roots and shoots of Kernza and the intercrop alfalfa (Medicago sativa) to form SOM in contrasting N management using continuously labeled 13C and 15N plant residues over 2 years. Our N management compared monocultures of Kernza that were unfertilized or fertilized, and a biculture of Kernza and alfalfa that was unfertilized. We hypothesized that the management and plant tissues with higher N may enhance mineral associated organic matter (MAOM) formation by alleviating microbial N-limitation and leading to enhanced microbial C use efficiency. Further, we hypothesized that root tissues would contribute to SOC sequestration primarily as occluded particulate organic matter (oPOM) due to their chemistry and interface with the soil matrix. We found that overall Kernza promoted SOM formation with 20% of roots and 12% of shoot stabilized after 27 months compared to 10% for alfalfa roots and shoots. Contrary to our expectations, there were no differences between the efficiency of MAOM formation of alfalfa vs. Kernza. The intercrop sustained similar C and N stocks to the fertilized treatment, though we found little evidence that N management influenced SOM formation. Using average aboveground biomass by N management to estimate soil C inputs and the rates of SOM formation from the incubation, we found that Kernza supports SOM formation rates between 0.11-0.18 ± 0.01 SE Mg C ha-1 yr-1 primarily from root C in surface soils in the ratio of 3:2 POM:MAOM. Roughly 20% of root tissues C incorporated into SOM after 27 months with 8% in MAOM and 7% in oPOM, implying that 15% of root tissue C may be stabilized in the soil. Legume intercrops can support cropping systems with minimal synthetic inputs, though they may not lead to enhanced SOCM formation even with comparable levels of productivity.These results are reported in a chapter of Dr. van der Pol's PhD Dissertation, and in a manuscript which is in submission to Frontiers. Objective 2: We addressed the 'soil carbon (C) dilemma' (Janzen 2006): "How can SOM be increased, while also increasing the release of nutrients that accompanies decomposition?" with an observational study of conventional, dryland wheat farmers in semi-arid Colorado and Nebraska. We tested whether incorporating legumes into a continuous rotation influences the form and amount of SOM as well as productivity in farms of the central Great Plains region of the U.S by contrasting three, no-till rotation systems: 1) conventional wheat-fallow; 2) continuous grain-only rotations, and 3) continuous grain rotations that incorporate a legume crop. We sampled on-farm fields and experimental agricultural research station plots that had received one of these rotations for at least eight years. We found that intensifying the rotation with continuous grains led to 1.5-fold increase in aggregate size but did not change SOC stocks. Incorporating a legume to the continuous grain rotation resulted in 1 Mg C ha-1 more SOC on average in surface soil compared to wheat-fallow rotations, thus providing a potential solution to the soil C dilemma. These findings are published in van der Pol et al., 2022, and are reported in a chapter of Dr. van der Pol, PhD dissertation Objective 3: We completed a laboratory incubation, which resulted in a MSc Thesis and findings are in a manuscript in submission for publication in Geoderma. Using 13C enriched structural and soluble plant inputs, we traced soil organic carbon (SOC) formation and stabilization in a lab incubation experiment in soils with differing levels of aggregation, and capacity to form aggregates after disturbance. Our results showed that soluble plant inputs contributed substantially to mineral associated organic carbon (MAOC) while structural plant inputs preferentially formed particulate organic carbon (POC). Moreover, soil type did not affect aggregate-occluded POC (oPOC) formation but did influence oPOC persistence after disturbance. We observed greater persistence of oPOC in the highly aggregated soil after disturbance in corroboration with our finding that disturbance of the low aggregation soil resulted in the highest CO2 mineralization of structural plant inputs. Interestingly, disturbance caused less mineralization of soluble inputs and more MAOC formation in the high aggregation soil suggesting that increased mineral surface area caused more efficient direct sorption of dissolved organic carbon (DOC). Regardless of disturbance, we observed that MAOC persisted in the high aggregation soil more than the low aggregation soil. Quantification of plant-derived C in the microbial biomass indicated that water soluble inputs are assimilated into microbial biomass more than structural plant inputs, thus incorporated into SOC with higher efficiency. Furthermore, water soluble inputs resulted in less loss of native SOC while structural plant inputs increased native SOC decomposition, generally. Lastly, the significant drop in plant-derived C in the microbial biomass overtime paired with water-soluble-derived MAOC persistence in the highly aggregated soils regardless of disturbance, illustrates the importance of microbial necromass in addition to direct DOC sorption for SOC stabilization as MAOC. Overall, this study provided mechanistic evidence suggesting that management for SOC accrual should promote structural plant C inputs in coarse textured soils with little capacity to form organo-mineral complexes, including large aggregates. Occasional disturbance can promote both POC and MAOC formation and stabilization in fine textured soils that have a high capacity to form organo-mineral complexes. Having established this mechanistic understanding, field studies are now required to very them these mechanisms under natural conditions and enable their broader generalization and incorporation in models.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Soil Health in Dry Lands: Pathways to rebuilding soil organic matter in dryland grain systems. Colorado Seed Growers Association Annual Meeting. Hilton Hotel, Fort Collins, CO, USA.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: van der Pol, L. Nester, B., Schlautman, B., Crews, T., Cotrufo, M.F. (2022) Perennial grain Kernza� fields have higher particulate organic carbon at depth than annual grain fields. Canadian Journal of Soil Science. https://doi.org/10.1139/cjss-2022-0026.
• Type: Journal Articles Status: Other Year Published: 2024 Citation: van der Pol, L., Crews, T., Cotrufo, F.M. Mechanisms of soil organic matter formation for perennial grain Kernza. in preparation for submission
• Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Rebecca Even, THE EFFECTS OF SOIL STRUCTURE ON SOIL ORGANIC MATTER FORMATION AND PERSISTENCE: A MECHANISTIC APPROACH, MSc Thesis, Colorado State University, 2022
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Even, R. & Cotrufo, M.F., The ability of soils to aggregate, more than the state of aggregation, promotes protected soil organic matter formation. In submission to Geoderma
• Type: Other Status: Other Year Published: 2022 Citation: Invited presentation: Regenerating soil organic matter for healthy and productive soils. Northwest Michigan Orchard and Vineyard Show.
• Type: Other Status: Other Year Published: 2022 Citation: The Epoch Times, October 24, 2022. Interviewed for: Soil in Danger: How Fixing Agricultural Soil Could Help the World
• Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Laura K van der Pol, GROWING DEEPER: PATHWAYS TO ENHANCING SOIL ORGANIC MATTER IN ANNUAL AND PERENNIAL DRYLAND GRAIN AGROECOSYSTEMS. PhD Dissertation, Colorado State University

Progress 03/01/21 to 02/28/22

Outputs
Target Audience:Our target audience are peer scientists, companies interested in regenerative agriculture, and producers. Results from this study have also been incorporated into seminar and lectures for graduate and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity to train and employ four undergraduate researchers this year in methods for soil sample processing and analysis. How have the results been disseminated to communities of interest?As the field days were cancelled due to the COVID19 pandemic, we presented the findings from Objective 1 at the international tri-society meeting (SSSA) held in Salt Lake City, UT in 2021, and incorporated them in brother conference meetings and seminar presentation from Dr. Cotrufo. We have also presented this work internally at CSU at a departmental seminar series. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Activities Now that the work from this project will be published imminently, we will write a brief summary of the key findings from this paper to disseminate on social media, with those involved in this study, and as a press release to news organizations such as Acres Magazine. Objective 2 Activities We will complete the sample analysis from the three harvests which primarily involves preparing and running them on the IRMS. We anticipate completing the data analysis and drafting a manuscript about these data for publication in the next six months. Objective 3 Activities This month we will complete all of the microbial biomass extractions for harvest 1 and harvest 2 (96 total samples). All H2 fractions will be run on the IRMS in addition to the freeze-dried DOM for both harvests. Microbial biomass extracts will be run on the TOC, freeze-dried, and run on the IRMS. We'll perform statistical analyses of all the data and write several papers (including my thesis). I will also present our current findings at the Front Range Student Ecology Symposium next week and defend in the summer. Both the GRA who contributed to this project, PhD candidate Laura van der Pol, an dMSc student Rebecca Even expect to graduate by the Fall 2022, and results from this project will be part of ther Dissertation and Thesis, respectively.

Impacts
What was accomplished under these goals? Objective 1: To meet this objective, we competed the model simulation of the rotations included in this study and submitted the manuscript for publication in the journal Agriculture, Ecosystems, & the Environment (AEE). After revisions, the manuscript was accepted for publication (2/4/22), and it is now published open access here: https://doi.org/10.1016/j.agee.2022.107906 Objective 2: In the past year, we completed the size-density fractionation of samples from harvest two and have run those samples on the isotope ratio mass spectrometer (IRMS). We completed the final (of three) harvests of soil collars from the field in Salina, KS in August 2021. These samples have been processed (sieved) and are being fractionated (70% complete). We are also currently processing the litter samples for analysis on the IRMS as well as quantifying microbial biomass. Frozen (-80 C) samples were also sent to a collaborator at the Los Alamos National Laboratory who is developing a method for protein stable isotope probing (protein-SIP-ML). This method uses high-throughput soil protein extraction coupled with stable isotope probing and machine-learning algorithms to identify microorganisms and their functions (protein) associated with functions of interest. This technique could potentially allow protein-metagenomic analysis with low levels of isotopic label, possibly also including 15N. As this is a novel technique, its efficacy still needs to be assessed. Objective 3: In September 2021, we completed our year-long incubation experiment and performed the second destructive harvest. All harvest 1 samples (day 22) have been fractionated, measured for water stable aggregates, and all fractions have been run on the IRMS to calculate 13C litter distribution among free particulate, occluded particulate, and the mineral associated organic matter fractions. All harvest 2 (day 366) soils have been fractionated, analyzed for water stable aggregates, and prepped to run on the IRMS. The respiration data is complete and compiled for cumulative total CO2 respiration and percent litter-derived soil respiration (13C respiration) for all treatments.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: van der Pol., L.K., Robertsona, A., Schipanski, M, Calderon, F.J, Wallenstein, M.D., Cotrufo, M.F. (2022) Addressing the soil carbon dilemma: Legumes in intensified rotations regenerate soil carbon while maintaining yields in semi-arid dryland wheat farms. Agriculture, Ecosystem and Environment.330,107906. https://doi.org/10.1016/j.agee.2022.107906
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cotrufo M.F. (2021) A coherent measurement-modeling approach to assess soil organic matter accrual and related co-benefits in agricultural land. AGU Annual meeting, New Orleans, December 13-17, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cotrufo M.F. (2021): Integrating measurements and modeling to advance understanding and forecasting of soil organic matter dynamics at scale. EMSL Integration meeting, October 4-7. 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: van der Pol, L., Robertson, A., Schipanski, M., Calderon, F., Wallenstein M.D., Cotrufo, M.F. (2021, Oct) Addressing the soil carbon dilemma: Legumes in intensified rotations regenerate soil carbon in semi-arid dryland wheat farms. International Soil Science Society of America Meeting, Salt Lake City, UT, USA. (Oral Presentation).

Progress 03/01/20 to 02/28/21

Outputs
Target Audience:We are preparing the results of Objective 1 for peer review publication. We will disseminate findings from this project at the next Field Day hosted by the USDA Long Term Agricultural Research Station at Akron, CO and to individual farmers and stations who participated in this study. Additionally, we have communicated with fellow students and scientists at Colorado State University and The Land Institute about on-going analyses for Objective 2. We also presented the general idea to virtual meetings to Industry stakeholders and local communities. Changes/Problems:Due to the lock down and subsequent limited access to the laboratory, our activities progressed slower than expected, and therefore we asked for a one-year extension. However, we managed to continue all the work, and we are confident we will deliver on all our goals by the new end date. What opportunities for training and professional development has the project provided?Despite the cessation of in-person activities for several months as well as temporary lab closure and severe restrictions on lab space availability, this project has provided opportunity for a student undergraduate researcher to complete an independent project in this reporting period. The student learned many foundational and complex soil analysis techniques including conducting a size and density soil fractionation as well as how stable isotopes can be used in ecological research. The student learned fundamental statistical analyses and computer programming in R Studio and presented her work as a poster (virtually) at CSU's Celebrate Undergraduate Research and Creativity (CURC) Symposium. Additionally, two student researchers were recently hired to assist with the sample processing who are keen to incorporate soil science in their future careers and are eager to have hands-on experience in a lab setting when so few opportunities are currently possible in person. The graduate students on this project have developed skills in seeking ethics approval for conducting (or not conducting) human subjects research, learned new and sophisticated lab techniques (e.g. isotopic analysis, PLFA), and developed more robust statistical, coding, and modelling expertise. How have the results been disseminated to communities of interest?Unfortunately, several events including the USDA Field Day in Akron, CO where we had planned to present were cancelled due to the onset of the COVID19 pandemic. Despite these limitations, we did share the on-site field data with each farmer who participated in Objective 1 of this research project and held one-on-one conversations with all who were interested. Presentations have otherwise thus far been mostly oriented to the research community this year. However, the Pi, Dr. Cotrufo, also gave several presentations to industrial and local communities. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Activities We plan to submit the manuscript for this objective to the journal Agriculture, Ecosystems, & Environment and present our findings at Field Day events that many in our study attend along with disseminate a short general-audience summary of our findings Objective 2 Activities During the next reporting period, we will complete the fractionation and PLFA analyses for harvest 1 and 2 as well as collect the third (final) harvest of the incubated litter from The Land Institute. We will also process and quantify the 13C/15N in the plant and litter samples, analyze the data and prepare for publication. Objective 3 Activities In the near future, we will analyze the H1 water stable aggregate data to test the hypothesis that plant inputs (primarily structural) can increase soil aggregation, especially in a disturbed soil with high aggregation potential. We will also fractionate H1 soils into 4 distinct SOM pools. Once fractionated, soil fractions will be homogenized and run on an EA-IRMS. This will allow us to trace SOM formation using the isotopic label and assess decomposition and stabilization of the litter inputs. The microbial analysis for H1 will also take place during this time. Gas measurements will continue until day 365 and so will analysis of 13C-CO2 to determine the contribution of litter to the CO2 flux. At the end of the incubation, we will repeat all soil analyses (water stable aggregates, fractionation, microbial analysis) on the H2 samples, analyze the full gas data set and prepare the data for publication.

Impacts
What was accomplished under these goals? For Objective 1, we have wrapped up data analysis for the observational field study and are nearing manuscript submission. We have added a modelling component to this experiment using the Microbial Efficiency Matrix Stabilization (MEMS v1.0) developed by A. Robertson et al. (2019). The model will allow us to extend our analysis of how a gradient of intensification (soil carbon input) under contrasting rotations and diversity of litter chemistry inputs (lower C:N from legumes) might influence soil organic matter in the study region if farmers continued or converted their management for a sustained period. Our work confirmed our hypothesis that including legume in crop rotation increase soil organic C, and that this C is protected by mineral association, at the same time of supporting higher overall yields, i.e., solving the "Soil C dilemma". For Objective 2, despite restrictions on travel and lab use due to COVID19, we completed the second harvest of field-incubated isotopically labelled litter from The Land Institute (Salina, KS). Prior to the lab closing in March of 2020, we had completed the fractionation and analysis on the EA-IRMS of 28 (of 87) samples from the first harvest. Since the lab closure during the pandemic, the instruments needed to analyze the soils fractions have not been operational. The samples from the second harvest were fully processed (bulk density, soil moisture quantified; 8mm and 2mm sieved; fresh samples frozen for PLFA), and we are in the process of fractionating the remainder of harvest 1 as well as the harvest 2 samples (93/174 complete). The graduate student leading analyses for this incubation project was trained in how to sample PLFAs and will begin measuring PLFAs in March 2021. For Objective 3, our initial start date was delayed drastically due to the pandemic. However, we still managed to begin in September and are now 156 days into the experiment. We designed the incubation in a factorial design with 2 soils types under 2 different levels of disturbance (undisturbed and disturbed), 3 litter treatments (control, structural, soluble), and 4 reps of each. We have two destructive harvests (n=48 for each harvest) in our incubation to assess the initial soil organic matter formation and then persistence. Our first harvest (H1) occurred 21 days into the incubation. All H1 soil respiration data has been plotted and analyzed preliminarily. Additionally, all H1 soils have all been processed, enriched structural plant material has been collected and weighed, and all soil samples have been tested for water stable aggregates using a wet-sieving method to separate aggregate size fractions. A subsample of each soil sample was also taken fresh for microbial analysis. Preliminary results from H1, seem to confirm our hypothesis in terms of how plant inputs impact aggregation. We also see differences in how structural and metabolic plant residues differ in decomposition and SOM contribution based on the respiration data. We are continuing the incubation and respiration measurements and have started fractionating the H1 soils.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Rebecca Even, Laura van der Pol, Francesca Cotrufo. Pathways of soil organic matter formation and protection in soil aggregates: a mechanistic approach. Front Range Student Ecology Symposium. Colorado State University, Fort Collins, CO. (Poster Presentation)
• Type: Other Status: Other Year Published: 2020 Citation: Cao, L., van der Pol, L. Preliminary Results Suggest Kernza Management Does Not Affect Mineral or Particulate Organic Matter Formation. Celebrate Undergraduate Research and Creativity Symposium. Colorado State University, Fort Collins, CO. (Poster Presentation)
• Type: Other Status: Other Year Published: 2020 Citation: Rebecca Even, Laura van der Pol, Francesca Cotrufo. Soil aggregates  the tiny homes of soil organic matter. Graduate Student Showcase. Colorado State University, Fort Collins, CO. (Poster Presentation)
• Type: Other Status: Other Year Published: 2021 Citation: M.F. Cotrufo, DIGGING DEEPER: Emerging Science and Case Studies on Soil-Carbon Sequestration. Webinar to Unilever.
• Type: Other Status: Other Year Published: 2021 Citation: M.F. Cotrufo. The 5 Ws (and 2 Hs) of Carbon Sequestration in Soils. Webinar to Broomfield County, CO.
• Type: Other Status: Other Year Published: 2020 Citation: M.F. Cotrufo, Carbon Sequestration in Soils. Stanford Carbon Management Workshop, Stanford, CA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: M. F. Cotrufo. Advancing understanding of soil organic matter to provide solutions to our wicked challenges. Invited Francis Clark Lecture, ASA-CSSA-SSSA virtual conference
• Type: Other Status: Other Year Published: 2020 Citation: M.F. Cotrufo. Regenerating Soil Health to Meet the 21st Century Challenges. Presentation to a workshop arranged by Nutrien.

Progress 03/01/19 to 02/29/20

Outputs
Target Audience:Our main target audience this year have been peer scientists. We have presented at several workshops and conferences ideas and results stemming from this project. In particular Dr. Cotrufo gave two invited talk at the ASA-CSSA-SSSA annual meeting, and a key note address at the SOM2019 meeting in Adelaide. Another target audience has been the broad public participating in the Prairie Festival. In fact Dr. Cotrufo was an invited speaker at the 2019 Land Institute Prairie Festival https://landinstitute.org/video-audio/soil-organic-matter-humanitys-true-capital/. Graduate students have also been a target audience, since Laura van Der Paul and Rebecca Even, the two graduate stdudent on this project, presented preliminary results from their studies at the Front Range Student Ecology Symposium in February 2020 at Colorado State University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the past year, we have retained an undergraduate who began sample processing on this project as well as trained two additional students in how to conduct soil analyses that include sample processing, operating the elemental analyzer, measuring soil texture, & pH as well as basics of data management and organization. These opportunities have guided the professional pursuits of these students who are exploring soil-related careers post-graduation. Additionally, we mentored one undergraduate through the process of developing a research question and conducting a short-term experiment and presenting results with others in the program. This student will go on to present a poster at a conference for undergraduates this spring. This fall, we began educating another undergraduate in the theory behind Objective 2 of this project. She will go on to conduct a research project beginning Spring 2020. in addition to the PhD student who began this project last year, we are supporting an additional Masters student to complete Objective 3 of this work. How have the results been disseminated to communities of interest?We have presented at several workshops and conferences ideas and results stemming from this project. In particular Dr. Cotrufo gave two invited talk at the ASA-CSSA-SSSA annual meeting, and a key note address at the SOM2019 meeting in Adelaide. She also was an invited speaker at the 2019 Land Institute Prairie Festival https://landinstitute.org/video-audio/soil-organic-matter-humanitys-true-capital/ Laura van Der Paul and rebecca Even, the two graduate students on this project, also presented preliminary results from their work at the Front Range Student Ecology Symposium in February 2020 at Colorado State University, and other events. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Activities There are a few analyses remaining to complete Objective 1, primarily completing the sequencing of the soil samples collected. We will select a protocol to use for sequencing, complete the quantification and PCR of these samples for analysis at the Microbiome Facility at Colorado State University. We expect to have a draft manuscript completed and submitted for publication on this first portion of the project, by early Summer. Additionally, once results have been summarized for each field site, we will disseminate a report to each farmer or station manager about our findings of their fields while also asking for a more detailed land-management history to inform our analyses. Objective 2 Activities We will complete analysis (PLFA and soil fractionation) of the first soil core harvest (n=54) of the litter incubation, and conduct the second (1 year) harvest in May 2020. Following we will start processing the samples collected in harvest 2 and the data from harvest 1. Objective 3 Activities We will conduct the laboratory experiment to identify pathways of soil organic matter formation and protection in soil aggregates. Throughout the experiment, CO2 concentration will be measured on an infrared gas analyzer (IRGA) to determine C mineralization rates. Gas samples will also be run on a mass spectrometer to obtain delta 13C-CO2. This will allow us to determine the soil-derived and the plant-derived CO2 flux. Two destructive harvests will take place. One at 14 days to assess fast SOM formations and priming effect and the other at 365 days, the end of the incubation, to assess litter-derived POM and MAOM stabilization.

Impacts
What was accomplished under these goals? We have almost completed all the laboratory analyses needed to answer Objective 1. Analyses of these data is underway. Laboratory analyses completed include measuring on four soil depths (0-100 cm, 114 samples): soil texture, pH, carbonate concentration, soil C and N. Additional analyses for the 0-10cm depth: DNA extraction for 16S and ITS1/2 sequencing; density and size fractionation and C & N quantification of 4 soil fractions; & water stable aggregates. The PhD student leading this portion of the project also received training in how to collect and analyze genomic sequencing data. To meet Objective 2, we set up a litter decomposition experiment with 13C and 15N labelled intermediate wheatgrass (aka Kernza, Thinopyrum intermedium) and alfalfa (Medicago sativa) at The Land Institute in Salina, KS in May 2019. We completed the first of three soil collar harvests in August 2019 and have completed the initial sample processing which includes weighing the soil cores, collecting a sub-sample for PLFA analysis, and 2mm sieving the air-dried sample for further analyses. To meet Objective 3, we have designed the laboratory incubation experiment, acquired and prepped all the soil and plant materials and run some preliminary tests and analyses. Soils (0-10 cm) were collected from the Konza Prairie Biological Station, KS and State Forest State Park, CO, to obtain a well aggregated and a naturally poorly aggregated soil sample. All bulk soils were gently 8mm sieved fresh, removing rocks, roots and any aboveground litter. For the aggregate disruption treatment, a subset of the soils were crushed dry to break aggregates without losing dissolved organic matter. Soils were also analyses for the texture, C and N content, pH and natural mean weight diameter, by conventional methods. For the incubation we will use 15N and 13C enriched structural plant component (SPC) and hot water extractable (HWE) materials from Kernza, a perennial wheat trademarked by The Land Institute. The bulk litter was oven-dried at 40C, ground on a Wiley mill and analyzed on the EA-IRMS for %C, %N, 13C atom%, and 15N atom%. HWE and SPC were separated by boiling ~60g of oven-dried kernza in 1.5 L of DI water at 105o F for an hour. The extract were filtered through 20 µm mesh and freeze-dried. The remaining SPC were rinsed with DI and oven-dried. Both HWE and SPC were analyzed on the EA-IRMS for %C, %N, 13C atom%, and 15N atom%. We are set to start the one-year incubation by the end of March.

Publications

Progress 03/01/18 to 02/28/19

Outputs
Target Audience:We have been in contact with all participants of our study which includes managers of agricultural research stations as well as privately owned wheat fields. Additionally, we have communicated with fellow students and scientists both at Colorado State University and abroad at the International Soils Conference meeting in January. Changes/Problems:This has been a very productive first year, and all the work planned was accomplished. The only change worth of notice is, as we explained in the report, that we will conduct the experiment to address Object 2, i.e. the dual labelled litterincubation, at the Land Instittue field site (Salina, Kansas)rather than at ARS research station in Akron, CO. What opportunities for training and professional development has the project provided?As part of this project, we trained three undergraduates in conducting basic soil processing and analyses as well as an undergraduate who learned new field techniques for measuring bulk density. Additionally, we mentored one undergraduate in the research process where she developed a research question, collected and analyzed data, and delivered a presentation at the end about her work. This project was able to hire a full-time research associate, allowing for deeper training and development of research staff at Colorado State University in soil analytical techniques. Through this project, we have sponsored a Ph.D. student who has begun study to become a soil scientist through the support of this project. How have the results been disseminated to communities of interest?As most of our samples are still being processed and analyzed, we have not focused our efforts on disseminating findings to stakeholders yet. Thus far our discussions have been with fellow scientists through poster presentations at conferences and scientific gatherings. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Activities To meet Objective 1 in the next year, we will finish processing and analyzing the 120 composited 0-10cm soil samples (divided into 4 depths). This will include completing the 2mm sieving of all samples by depth (480 samples), completing the water stable aggregate and size and density fractionation of 0-10cm depths, 16S and ITS1 PCR sequencing of microbial communities in the 0-10cm depth, and measuring total carbon & nitrogen, bulk density, soil pH, and texture for all soil depths. Once analyses are completed, we will send an individualized report to each grower or research institution participating in this study to share with them the soil properties we identified at their site. We will also perform statistical analyses to assess the role of diversification and intensification on established farm rotations as described in Objective 1. Additionally, we will interview land managers who are part of this project for their land use history and practices that will be included in our analyses for this study. Objective 2 Activities The activities described above will inform Objective 2, but we will use the isotopically labelled Kernza and alfalfa to begin a two-year decomposition experiment to further elucidate Objective 2. This spring, we will place 176 soil collars in the experimental plots at The Land Institute, collect initial soil samples, and harvest soils at the 1-month and 6-month time-points. These soils will be analyzed for PLFAs, soil C and N by soil fraction, water-stable aggregate stability, as well as root biomass as applicable. Objective 3 Activities Based on the results from our water-stable aggregate measurements on the field-soils collected in August 2018, we will select a site with high aggregation to collect soils needed to meet Objective 3. At a convenient time for the land manager, we will travel to that site and collect the soils to address the role of aggregation in soil carbon and nitrogen processing and stabilization and soil microbial community.

Impacts
What was accomplished under these goals? We have collected the samples needed to answer Objective 1 in this study and expect to have the results analyzed by summer 2019. Additionally, we have located a field site and grown the isotopically labeled litter for the mechanistic studies essential to meeting Objective 2 and 3. To meet Objective 1 above and inform Objectives 2 and 3, we collected 120 0-100cm soil samples (composites of 3 soil cores, divided into four depths: 0-10, 10-30, 30-50, and 50+ cm) from 15 field sites in eastern Colorado and western Nebraska. We have completed the initial soil processing which included 8mm fresh-soil sieving where we measured soil moisture and bulk density and removed rocks and plant debris, preserving a sample for microbial community analyses (-80? storage), and air-drying the soils for further processing. We have begun measuring water-stable aggregates on the 0-10 cm soils and are well-underway 2mm sieving the air-dry soil samples for further analyses. Additionally, we conducted a study of rhizosphere and bulk soils at one of our field sites (USDA ARS in Akron, CO) where we uncovered evidence for synergistic relationships between the rhizosphere microbial community and plant growth as well as possible support for the major framework tested by this work. These results were presented at the International Soils Meeting in San Diego, CA. As a result of our study in Akron, CO, we determined that the variability of the soil texture and pH at that site may confound any effects for our labelled decomposition project to address Objective 2. We thus found an alternative site that is not only suitable for our study but which may further our understanding of emerging practices to incorporate perennial plants into grain systems. This new site is based in Salina, KS and is a partnership with The Land Institute which has domesticated a perennial intermediate wheatgrass called Kernza. As this is a perennial system, our focus on testing diversification and intensification effects on soil carbon will shift from being a gradient in time (annual system) to a gradient in space (perennial system) with row spacing and intercropping being the treatment groups rather than rotations. To begin this mechanistic study of organic matter formation, we have grown 13C and 15N isotopically labeled Kernza and alfalfa in the Plant Growth Facilities at Colorado State University. The plants have been harvested and air-dried on January 15, 2018 and their isotopic ratios and mass is under measurement.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: van der Pol, L., Even, R., Calderon, F., Schipanski, M. Wallenstein, M., Cotrufo, M.F. Drivers and Indicators of Soil Health in Semi-Arid Agroecosystems: Addressing the ⿿soil carbon dilemma⿝ through the lens of microbes and soil structure. USDA NIFA Water and Soils PD Meeting Oct 1-3, 2018, University of Delaware.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: van der Pol, L., Calderon, F., Schipanski, M. Wallenstein, M., Cotrufo, M.F. (2018) Can cultivating connectivity solve the soil carbon dilemma? Colorado State University Graduate Student Showcase, Fort Collins, CO, USA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: van der Pol, L., Calderon, F., Schipanski, M. Wallenstein, M., Cotrufo, M.F. (2019) Synergy between crop growth and microbial activity in the rhizosphere but not bulk soils. Soil Science Society of America International Meeting, San Diego, CA, USA.