MICROBIAL CONTRIBUTION TO BUILDING AND STABILIZING SOIL ORGANIC MATTER UNDER LONG-TERM CROP MANAGEMENT PRACTICES IN AGROECOSYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022313
• Grant No.: 2020-67019-31160
• Cumulative Award Amt.: $499,744.00
• Proposal No.: 2019-06567
• Project Start Date: Apr 15, 2020
• Project End Date: Apr 14, 2025
• Grant Year: 2020
• Program Code: [A1401]- Foundational Program: Soil Health

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
SOIL SCIENCE

Non Technical Summary
Enhancing soil organic matter (SOM) level is essential to restoring degraded soils, promoting higher agronomic productivity, and mitigating climate change. Recent conceptual and analytical advancement indicate that the microbial pathway of SOM formation and stabilization might be the primary mechanism for C accumulation. However, our ability to adopt management practices to improve SOM is still hindered by the lack of evidence of how microbial SOM dynamics respond to agricultural management at large scale. This project aims to address this knowledge gap and study the impact of crop management strategies on the microbially-regulated SOM dynamics across a range of agroecosystems in the United States. We hypothesize that including legumes in the rotation as cash or cover crops will increase the quality of plant-C input (lower C:N ratio) therefore lead to greater microbial C use efficiency and greater microbial necromass accumulation, hence increase the SOM content. By employing a combination of laboratory and field-based analyses at three long-term trials in Wisconsin, Iowa, and Pennsylvania, we aim to evaluate how long-term crop management strategies influence the structural and functional potential of soil microbial communities, SOM quality and stability, and the ability of SOM to supply nutrients. Improved knowledge on soil C accumulation generated by this research directly addresses the goal of investigating the effects of management practices on soil microbial community's function and their contribution to healthy soils and sustainable agroecosystems under Program Area Priority A1401-Soil Health. The findings can also facilitate the adoption of best management strategies to maximize productivity and profitability, and sustainability of agroecosystems.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

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

soil health

soil organic health

Goals / Objectives
Traditional theory suggests the formation of SOM is a result of chemical complexity: recalcitrant plant inputs persist in the soil while easily decomposable plant inputs are respired as CO2. However, large inputs of recalcitrant crop residues (e.g., corn residues) in intensive production systems are often not associated with proportional gains in SOC (Leifeld and Fuhrer, 2010; Sanford et al., 2012). Instead, emerging experimental and theoretical evidence shows that stable SOM in organo-mineral associations and microaggregates are dominated with chemically labile rather than complex compounds; microbial products might be the main precursors of stable SOC by promoting aggregation and through strong chemical bonding to the mineral matrix (Cotrufo et al., 2013; Kallenbach et al., 2015; Liang et al., 2013 and 2016). Therefore, the efficiency of microbial biomass production, as well as the subsequent stabilization of microbial necromass, may play a pivotal role in soil C sequestration. In the grain-based cropping systems of United States, management practices that can increase the quality of C input by adding low C:N ratio legumes in crop rotation or applying manure have been found to increase SOM content and reduce fertilizer need (Karlen et al., 2006; Liebman et al., 2008; Coulter et al., 2011; Jokela et al., 2011). However, it is still not well understood if the increased SOM content is the direct result of more efficient microbial biomass production and greater microbial necromass accumulation under these practices.The overall goal of this project is to understand the interactions between crop management practices and microbial communities in facilitating soil C turnover in a range of agroecosystems of the United States.To meet this goal, we have identified four supporting project objectives:Objective 1. Using long-term replicated field experiments in three states with different soil types and environmental conditions, determine the impact of different crop management strategies on quality and quantity of C inputs, and test the hypothesis that including legumes in crop rotation and/or applying manure can improve both quantity and quality of C inputs in cropping systems at a broad scale.Objective 2. Using in-situ and laboratory incubations, determine the impact of different crop management practices on the size, structure, and functions of soil microbial community, and test hypothesis that including legumes in the crop rotation and/or applying manure can increase microbial CUE; Identify the relationships between CUE, microbial community structure and quality of C input at a large scale.Objective 3. Determine the impacts of crop management practices on the accumulation of microbial necromass; Identify the microbial modulator of SOC storage by evaluating the relationships between microbial CUE/necromass and SOC stored in stable SOM fractions (mineral associated C).Objective 4. Determine the impact of the crop management practices on SOM quality, stability and the capacity of SOM to supply nutrient, and evaluate if greater microbial necromass accumulation and SOM stability also lead to greater nutrient supply.Research will be conducted in cropping systems across different states of the United States (Wisconsin, Iowa, and Pennsylvania) to evaluate how different cropping systems and agricultural management affect the quantity and quality of C input, and the impacts on microbial community structure and functions, microbial necromass accumulation, and SOM stability. By identifying the interactions between cropping systems, management practices, soil microbial physiology and functions, and SOC storage, this work fundamentally seeks to understand the role that crop management strategies play in SOC accumulation. These findings will provide a strong framework to determine the impact of crop management practices on soil C and soil health and will provide justification of future agroecological intensification (green manure, dairy manure, cover cropping, and livestock integration). Research findings can facilitate the adoption of best management practices to maximize productivity and profitability, and the sustainability of agroecosystems.

Project Methods
Objective 1:Praticulate Organic Matter (POM): We will fractionate soil organic matter (SOM) into a fast-cycling, plant-derived particulate organic matter (POM) pool, and a slow-cycling, microbe-derived mineral-associated organic matter (MAOM) pool, using the method described in Bradford et al. (2008). Briefly, duplicate soil samples (10 g of air-dried soil) from each plot will be dispersed with 30 mL sodium hexametaphosphate (NaHMP) via shaking (18 h) and then passed through a 53 μm sieve. Material <53 μm is considered MAOM and material >53 μm is considered POM.Objective 2:Phospholipid Fatty Acids (PLFA):The biomass and community structure of soil microbial community will be assessed by the phospholipid fatty acids (PLFA) analysis. Briefly, membrane lipids of soil microbes will be extracted, purified, and then separated by silicic acid chromatography as outlined by Herzberger et al. (2014). Total nmol lipid g soil-1 is used as an index of microbial biomass and to calculate fungi:bacteria ratio (Balser and Firestone, 2005). The relative contribution of specific lipid biomarkers to the microbial community is determined by calculating the moles of a given lipid/total moles lipid per sample (mol%).CUE (18O-H2O method): The microbial CUE will be determined based on the incorporation of 18O from 18O-labeled H2O into microbial genomic DNA following the procedures described by Spohn et al. (2016). Briefly, 300 mg of pre-incubated soil will be weighed into 2 ml vials. H218O (97.0 18O atom%,) will be added to one of the replicates to reach 20.0 atom% of 18O in the final soil water. Then, the open vials containing the amended soils will be immediately transferred into 50 ml incubation containers sealed with a crimp cap and incubated at 25°C for 24 hours. The 2 ml vials will then be taken out for soil DNA extraction. A gas sample will be taken to determine the CO2 concentration using a gas chromatography. The soil DNA concentration in each extract will quantified using a NanoDrop spectrophotometer (Thermo Scientific, USA). The DNA extract will be dried in silver capsules at 60°C, prior to the determination of the 18O abundance and the total O content by an isotope ratio mass spectrometer coupled with an elemental analyzer. The amount of DNA produced during the incubation will be transformed into the amount of MBC produced using linear regression equation between the concentration of DNA and MBC. Microbial growth and microbial respiration flux will be determined using the method developed by Spohn et al. (2016).13C assimilation: An in-situ isotope tracer experiment will be established using 13C-labeled glucose and phenol to study the assimilation of 13C into microbial biomass and mineral-associated SOC pools. We will follow the method developed by Kallenbach et al. (2015). In early season (April), we will insert 7 PVC tubes (10-cm diameter), open at the top and bottom, 10 cm into the soil in each of the plots. Within each plot, 3 tubes will receive 13C-labeled phenol, 3 will receive 13C-labeled glucose, and 1 will serve as a control that only receives water. 99-atom% 13C-glucose or 13C-phenol mixed with deionized water will be syringe-injected to a 5-cm depth into each core.Objective 3:Amino sugars:We will also assess the relative contribution of the fungal and bacterial necromass to SOC accumulation, which are represented by ratios of amino sugars to SOC (Amelung et al., 2001; Liang et al., 2013). We will determine the content of detectable bulk soil amino sugars including glucosamine (GluN), which is predominantly derived from fungal cell walls and muramic acid (MurA), which is unique to bacteria, as described by Zhang and Amelung (1996). Soil samples will be hydrolyzed with 6 M HCl for 8 hr at 105°C, filtered, dried using a rotary evaporator, and redissolved in deionized water. After neutralizing with KOH, the samples will be centrifuged and the supernatant will be freeze-dried, after which amino sugars will be washed out from the residues with methanol and centrifuged to remove the salts. Derivatization reagents will be added to the dried residue before heating at 75-80°C to transform amino sugars into aldononitrile derivatives, which will then be extracted with 1.5 ml dichloromethane from the aqueous solution. The amino sugar derivatives are finally separated on a gas chromatograph. The ratio of GluN/MurA will be calculated to elucidate relative dominance of fungal to bacterial necromass in SOC.Objective 4:DRIFTS:The stability of SOM will also be determined by the Diffuse Reflectance Mid-Infrared Fourier Transform Spectroscopy (DRIFTS) analysis. For this analysis the finely ground soil subsamples will be diluted with 97% KBr to eliminate scatter light intensity. The mixed samples (soil:KBr = 1:80) will then be homogenized by further grinding and scanned 64 times per spectrum from 4,000 to 400 cm-1 with a resolution of 4 cm-1 in reflectance mode, using a Fourier transform spectrometer. We will calculate the relative peak areas of aromatic C=C, ketone and quinone C=O, and/or amide N=H at 1,620 cm-1 (rA1620) and aliphatic C-H at 2,930 cm-1 (rA2930) using a tangential baseline method described by Demyan et al. (2012).Enzymes: The activities of the oxidative enzymes, phenol oxidase (PPO) and peroxidase (PER), will be studied to assess the potential of SOM loss under different management practices. Studies show that the activities of the oxidative enzymes are negatively correlated with SOM content, and higher activities of these enzymes suggest larger potential of SOM loss. PPO and PER will be measured using L-DOPA as the substrate. Microplates will be incubated in the dark at 20°C for four hours. PPO and PER assays will be read at 460-nm absorbance after transferring the supernatant to a fresh microplate to avoid absorbance interference by soil particles. Enzyme activity will be calculated as the amount of substrate cleaved during the incubation.Mineralizable N: The potentially mineralizable N (PMN) is biological measure of the soil's capacity to supply N through the mineralization of soil organic N reserves. PMN will be measured in the laboratory on soils by measuring ammonium N produced in soil incubated under anaerobic conditions for 7 days (Waring and Bremner, 1964).Evaluation: A detailed sampling and analysis plan will be followed to ensure completion of all sampling and analysis.

Progress 04/15/20 to 04/14/25

Outputs
Target Audience:The target audience of this project is research scientists, extension educators, and crop consultants. The primary audience is research scientists, as this work will reveal mechanisms and controls on soil carbon cycling and storage, and will be useful in both conceptual and empirical modeling of soil C. While this research is focused on mechanisms of carbon cycling and storage in surface soils, the results will be of interest to those aiding on-farm decision making, as specific crop practices are assessed and connected to real gains in soil C. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Tanner Judd (PhD graduate student) had the opportunity to gain presentation and extension skills through the following events: Soil Science Society of America Conference (poster & oral presentations), FarmFirst Dairy Co-op board (oral presentation), UW Department of Soil Science seminar (oral presentation), UW-CALS Board of Visitors Sustainability panel (oral presentation), the Professional Dairy Producers of Wisconsin Business Meeting (invited oral presentation), and the North Central Soil Fertility Conference (poster presentation). He has attended three scientific conferences and three extension conferences. He has also had the opportunity to write a SARE graduate student grant to advance his research in this area and was funded. He also had the opportunity to serve as a teaching assistant for Soil Science 101 (Forum on the Environment) in the spring of 2023. How have the results been disseminated to communities of interest?The results have been presented four times at scientific conferences or seminars: 1. Microbial Contribution to C Turnover and Stabilization in Long-Term Cropping Systems Under Varying Management Practices (November 8th, 2022) Tri-Societies Conference, Baltimore, Maryland 2. Microbial Contribution to C Turnover and Stabilization Altered by Long-Term Cropping System Diversification and Perenniality (October 31st, 2023) Tri-Societies Conference, St. Louis, Missouri 3. How Do Microbes Contribute to Soil Organic Matter Persistence in Rotationally Diverse Cropping Systems in the North Central US? (November 12th, 2024) Tri-Societies Conference, San Antonio, Texas 4. UW-Madison, Department of Soil Science seminar, Carbon use efficiency and dairy production, (October 4th, 2023) The results have been presented at: 1. FarmFirst Dairy Co-op board presentation, July 2023, "Soil health and dairy production systems", 19 attendees (part of a bigger research tour organized by the Dairy Innovation Hub). 2. UW-CALS Board of Visitors Sustainability Panel, October 2023, "Sustainability and Dairy Production", 24 attendees (the Board of Visitors committee), an invited presentation by the UW-CALS Associate Dean of Research 3. Professional Dairy Producers of Producers Business meeting, March 2024, "Cover Crops and Manure - Partners in Conservation" 4. Dairy Signal (podcast of the Professional Dairy Producers of Wisconsin), October 2023, "Soil Health and Dairy Production", 140 attendees 5. Badger Crop & Soil updates, November 30 & December 2023, "Soil Health and On-Farm Trials", 400 attendees 6. Arlington Soil & Agronomy Field Day, August 2023, "Cover Crops...Like Tech But Better", 150 attendees 7. Global Conversations webinar (sponsored by CALS Global), February 2024, "Production vs. Conservation in the Dairy State" 8. Ag Source Conference, March 2024, "Soil Health and Dairy Production", invited talk by Ag Source soil test lab 9. Foremost Farms/UW-CALS board presentation (July 2024) "Interdisciplinary Research and Soil Conservation", invited presentation, 21 attendees, (part of a bigger research tour organized by UW-CALS and Dairy Innovation Hub). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplishments: Field sampling was conducted in 2022 at the Wisconsin Integrated Cropping Systems Trial (WICST) (Arlington, WI), and in 2023 at both WICST and the Marsden Long-Term Rotational Study (Ames, IA). Associated laboratory analyses are nearly complete and all samples for pending analyses are in the pipeline at service labs. Tanner Judd is the PhD student on this project. Sampling: In 2022, ten 30 cm soil cores were taken from replicate plots of six cropping systems at WICST at two times across the growing season (July and September 2022). In 2023, ten 30 cm soil cores were taken from crop rotation systems at WICST as well as the Marsden Plots at Iowa State University, also at two sampling times (July and September 2023). Sampling similar cropping systems on similar soil types allows for more generalizable inferences into plant-soil-microbial mechanisms mediating C storage in Mollisol soil. The soil cores were split into two depths (0-15, 15-30cm) and homogenized to create one sample / depth / system. For 2022, 96 total soil samples were collected and analyzed (1 site x 6 cropping systems x 2 soil depths x 4 blocks x 2 time points). For 2023, 64 total samples were collected and analyzed (2 sites x 2 cropping systems x 2 soil depths x 4 blocks x 2 sampling times). Analysis: Analyses completed across both sample times in 2022 and 2023 include carbon use efficiency (using H2 18O), microbial group biomass by phospholipid fatty acid analysis (PLFA), bacterial and fungal necromass by amino sugar assays, soil organic matter fractionation in to "fast" and "slow" cycling carbon, total CN combustion analysis of whole and fractionated soil, and soil organic matter complexity by mid-infrared spectroscopy. Statistical analysis with linear mixed effects models has been completed on 2022 data and a publication was submitted for review in November 2024. As of 4/2025 it is in revision. In this manuscript, our group determined that cropping system-specific effects on soil C properties were consistent throughout the mid-season and late season and were greatest in the surface soils and the cropping system effects were more subtle or absent in the deeper soil. For example, POM C:N, an indicator of plant input quality, was the lowest under pasture in the surface soil, whereas the C:N under most cropping systems were similar in deeper soils. However, cropping system effects on microbial properties were less depth- or time- dependent. For example, a system x depth interaction was only observed for fungal biomass and no other indicators as estimated by PLFA. Similarly, microbial CUE and growth rate were affected by cropping systems similarly across time. Strong correlations were also observed between measured microbial and soil C properties. A second manuscript with 2023 data is currently in preparation and will be submitted by June 2025. In this manuscript, we compare the time and depth dependency of microbial and soil properties that mediate persistent C in long-term cropping system experiments in Wisconsin and Iowa, both of which on Mollisol soil. We determined that extended 4-year forage-based rotations supported greater soil microbial activity and abundance, compared to 2-year grain-based rotations, however system effects differed by both location and sampling time. For example, a system x location interaction for mineralizable carbon (MinC), an indicator of microbial respiration, and Fungi:Bacteria (F:B) were only observed at the Iowa location. There were also significant effects of sampling time on nearly all measured microbial properties including microbial growth rates (MGR), Total PLFA, and MinC, with measurements being greater at mid-season than late-season sampling. However, a system x time interaction for MGR indicated that all system-level differences found at mid-season were dampened at late-season. Impact statement based on project goal Over the short-term, there has been a change in knowledge. Scientists and land-owners now understand why certain management practices may not always lead to carbon gains in soil. Scientists specifically also have learned that there is a big difference in carbon use efficiency measurements based on 13C or 18O methodologies. Additional research will be needed into why the different isotopic method would produce different results. Lastly, scientists, farmers, and agronomics know about the large seasonal differences in biological measurements in the soil, which would encourage soil health sampling in late spring/early summertime periods. Based on this knowledge, farmers and land-owners can make changes in practice. This will entail explore opportunities to reduce tillage and to increase carbon additions to soil through cover cropping. Even dairy-based systems will need further reductions in tillage and increases in carbon for changes in CUE to occur. In the end, this work will influence the change of condition of Wisconsin soils having greater soil C and soil health. Or at minimum, it will help maintain soil C contents in soil, preventing a further reduction.

Publications

• Type: Other Status: Under Review Year Published: 2025 Citation: Judd, T.C., Ruark, M.D., Rui, Y., Sanford, G.R., Freedman, Z.B., 2024. Mechanisms of Soil Organic Matter Persistence Vary Across Time and Soil Depth in Long-Term Cropping Systems of the North Central US. (publication under review- submitted November 2024)
• Type: Other Status: Other Year Published: 2025 Citation: Judd, T.C., Ruark, M. D., Rui, Y., McDaniel, M. D., Sanford, G.R., & Freedman, Z.B., 2025. How Do Microbes Contribute to Soil Organic Matter Persistence in Rotationally Diverse Cropping Systems in the North Central US? (manuscript in preparation, with an anticipated submission date of May 1, 2025)

Progress 04/15/23 to 04/14/24

Outputs
Target Audience:The target audience of this project is research scientists, extension educators, and crop consultants. The primary audience is research scientists, as this work will reveal mechanisms and controls on soil carbon cycling and storage, and will be useful in both conceptual and empirical modeling of soil C. While this research is focused on mechanisms of carbon cycling and storage in surface soils, the results will be of interest to those aiding in on-farm decision making, as specific crop practices are assessed and connected to real gains in soil C. Changes/Problems:After years of significant COVID-related delays, this current reporting period has been smooth. It was a year of results being obtained and manuscripts being prepared. We do not anticipate any issues during the no-cost extension. What opportunities for training and professional development has the project provided?Professional training for Tanner Judd who is a PhD student on this project: Presentations- Farm First Dairy Co-op board presentation (July 2023) UW Soil Science Seminar presentation (October 2023) UW-CALS Board of Visitors Sustainability panel presentation (October 2023) Oral presentation- Tri-Societies meeting (October 2023) Professional Dairy Producers of Wisconsin Presentation (March 2024) Professional Dairy Producers of Wisconsin Webinar (April 2024) Conferences Attended- Tri-Societies Meeting (Fall 2023) Discovery Farms/Wisconsin Water and Soil Health Conference (December 2023) Professional Dairy Producers Business Conference (March 2024) Grants- Awarded NC-SARE Graduate Student Grant (Summer 2023) UW Center for Integrated Agricultural Systems mini grant (April 2024) Other professional development- Teaching assistant for SOIL 101: Forum on the Environment (Spring 2023) How have the results been disseminated to communities of interest?The results have presented two times at scientific conferences or seminar: Microbial Contribution to C Turnover and Stabilization Altered by Long-Term Cropping System Diversification and Perenniality (October 31st 2023) UW-Madison, Department of Soil Science seminar, Carbon use efficiency and dairy production, 2023 October 4 The results have been presented at: 1. Farm First Dairy Co-op board presentation, 2023 July, "Soil health and dairy production systems", 19 attendees (part of a bigger research tour organized by the Dairy Innovation Hub). 2. UW-CALS Board of Visitors Sustainability panel, 2023 October), "Sustainability and dairy production", 24 attendees (the Board of Visitors committee), an invited presentation by the UW-CALS Associate Dean of Research 3. Professional Dairy Producers of Producers Business meeting, 2024 March 13, "Cover crops and manure - partners in conservation" 4. Dairy Signal (podcast of the Professional Dairy Producers of Wisconsin), 2023 October 24, "Soil health and dairy production", 140 attendees 5. Badger Crop & Soil updates, 2023 November 30 & 2023 December 2, "Soil health and on-farm trials", 400 attendees 6. Arlington Soil & Agronomy Field Day, 2023 August 23, "Cover crops...like tech but better", 150 attendees 7. Global Conversations webinar (sponsored by CALS Global), 15 February 2024, "Production vs. conservation in the dairy state" 8. Ag Source Conference, 12 March 2024, "Soil health and dairy production", invited talk by Ag Source soil test lab 9. Dairy Signal (podcast of the Professional Dairy Producers of Wisconsin), 2024 April, "Cover Crops and Manure", 140 attendees What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan on completing the 2023 data analysis and publishing at least two manuscripts in peer-reviewed journals.

Impacts
What was accomplished under these goals? Field sampling was conducted in summer 2023 at the Wisconsin Integrated Cropping Systems Trial (WICST) (Arlington, WI) and the Marsden Long-Term Rotational Study (Ames, IA). Associated laboratory analyses are nearly complete and all samples for pending analyses are in the pipeline at service labs. Analysis is nearly complete from the 2022 field season and the remaining samples are in the pipeline and relevant service labs. We expect to have the complete 2022 dataset in hand in early summer 2024 and will complete the analysis and manuscript preparation as soon as possible after completing the dataset.Tanner Judd is the PhD student on this project. Sampling: In summer 2023, ten 30 cm soil cores were taken from crop rotation systems at WICST as well as the Marsden Plots at Iowa State University, at two sampling times (July and September). Sampling similar cropping systems on similar soil types allows for more generalizable inferences into plant-soil-microbial mechanisms mediating C storage in Mollisol soil. The soil cores were split into two depths (0-15, 15-30cm) and homogenized to create one sample / depth / system. For 2022, 96 total soil samples were collected and analyzed (1 site x 6 cropping systems x 2 soil depths x 4 blocks x 2 time points). For the 2023 field season, 64 total samples were collected and analyzed (2 sites x 2 cropping systems x 2 soil depths x 4 blocks x 2 sampling times). Analysis: Analyses completed across both sample times include carbon use efficiency (using H218O), microbial group biomass by phospholipid fatty acid analysis (PLFA), bacterial and fungal necromass by amino sugar assays, soil organic matter fractionation in to "fast" and "slow" cycling carbon, total CN combustion analysis of whole and fractionated soil, and soil organic matter complexity by mid-infrared spectroscopy. Statistical analysis with linear mixed effects models on 2022 data has been completed and a publication is being prepped for submission. A second manuscript with 2023 data will then be produced and submitted by April 2025.

Publications

Progress 04/15/22 to 04/14/23

Outputs
Target Audience:The target audience of this project is research scientists, extension educators, and crop consultants. The primary audience is research scientists, as this work will reveal mechanisms and controls on soil carbon cycling and storage, and will be useful in both conceptual and empirical modeling of soil C. While this research is focused on mechanisms of carbon cycling and storage in surface soils, the results will be of interest to those aiding in on-farm decision making, as specific crop practices are assessed and connected to real gains in soil C. Changes/Problems:This past year has been relatively smooth in terms of project management, with all data being collected and data being analyzed and reported for publication. We do not anticipate any issues during the final no-cost extension year of the project. What opportunities for training and professional development has the project provided?Tanner Judd (PhD graduate student) had the opportunity to gain presentation and extension skills through the following events: Soil Science Society of America Conference (poster & oral presentation), FarmFirst Dairy Co-op board (oral presentation), UW Department of Soil Science seminar (oral presentation), UW-CALS Board of Visitors Sustainability panel (oral presentation), and the Professional Dairy Producers of Wisconsin Business Meeting (invited oral presentation). He has attended two scientific conferences and two Extension conferences. He has also had the opportunity to write a SARE graduate student grant to advance his research in this area and was funded. He has also had the opportunity to serve as a teaching assistant for Soil Science 101 (Forum on the Environment) in the summer of 2023. How have the results been disseminated to communities of interest?The results have presented two times at scientific conferences or seminar: 1.Microbial Contribution to C Turnover and Stabilization in Long-Term Cropping Systems Under Varying Management Practices (November 8th 2022) 2. Microbial Contribution to C Turnover and Stabilization Altered By Long-Term Cropping System Diversification and Perenniality (October 31st 2023) 3. UW-Madison, Department of Soil Science seminar, Carbon use efficiency and dairy production, 2023 October 4 The results have been presented at: 1. FarmFirst Dairy Co-op board presentation, 2023 July X, "Soil health and dairy production systems", 19 attendees (part of a bigger research tour organized by the Dairy Innovation Hub). 2. UW-CALS Board of Visitors Sustainability panel, 2023 October X), "Sustainability and dairy production", 24 attendees (the Board of Visitors committee), an invited presentation by the UW-CALS Associate Dean of Research 3. Professional Dairy Producers of Producers Business meeting, 2024 March 13, "Cover crops and manure - partners in conservation" 4. Dairy Signal (podcast of the Professional Dairy Producers of Wisconsin), 2023 October 24, "Soil health and dairy production", 140 attendees 5. Badger Crop & Soil updates, 2023 November 30 & 2023 December 2, "Soil health and on-farm trials", 400 attendees 6. Arlington Soil & Agronomy Field Day, 2023 August 23, "Cover crops...like tech but better", 150 attendees 7. Global Conversations webinar (sponsored by CALS Global), 15 February 2024, "Production vs. conservation in the dairy state" 8. Ag Source Conference, 12 March 2024, "Soil health and dairy production", invited talkby Ag Source soil test lab. What do you plan to do during the next reporting period to accomplish the goals?Spring 2024 · Based on the number of samples that need to be analyzed and the labor associated with each method, we estimate it will take us until Spring of 2024 to complete all analysis. Spring & Summer 2024 · Complete and promulgate Annual Report of project activities, findings, and accomplishments. · Analyze the results and develop drafts of tables and figures · Field sampling at WICST and soil analysis Fall 2024 - Spring 2025 · Draft and submit two publications (#1, two years of WICST; #2, WICST vs. Marsden) · Present findings at grower and professional conferences and extension meetings; distribute outreach materials. · Graduate student completes PhD

Impacts
What was accomplished under these goals? 1.Accomplishments: Field sampling was conducted in 2022 at the Wisconsin Integrated Cropping Systems Trial (WICST) (Arlington, WI), and in 2023 at both WICST and the Marsden Long-Term Rotational Study (Ames, IA). Associated laboratory analyses are nearly complete and all samples for pending analyses are in the pipeline at service labs. Tanner Judd is the PhD student on this project. Sampling: In 2022, ten 30 cm soil cores were taken from replicate plots of six cropping systems at WICST at two times across the growing season (July and September 2022). In 2023, ten 30 cm soil cores were taken from crop rotation systems at WICST as well as the Marsden Plots at Iowa State University, also at two sampling times (July and September 2023). Sampling similar cropping systems on similar soil types allows for more generalizable inferences into plant-soil-microbial mechanisms mediating C storage in Mollisol soil. The soil cores were split into two depths (0-15, 15-30cm) and homogenized to create one sample / depth / system. For 2022, 96 total soil samples were collected and analyzed (1 site x 6 cropping systems x 2 soil depths x 4 blocks x 2 time points). For 2023, 64 total samples were collected and analyzed (2 sites x 2 cropping systems x 2 soil depths x 4 blocks x 2 sampling times). Analysis: Analyses completed across both sample times in 2022 and 2023 include carbon use efficiency (using H218O), microbial group biomass by phospholipid fatty acid analysis (PLFA), bacterial and fungal necromass by amino sugar assays, soil organic matter fractionation in to "fast" and "slow" cycling carbon, total CN combustion analysis of whole and fractionated soil, and soil organic matter complexity by mid-infrared spectroscopy. Statistical analysis with linear mixed effects models has been completed on 2022 data and a publication is being prepped for submission. A second manuscript with 2023 data will then be produced and submitted by April 2025.

Publications

Progress 04/15/21 to 04/14/22

Outputs
Target Audience:The target audience of this project is research scientists, extension educators, and crop consultants. The primary audience is research scientists, as this work will reveal mechanisms and controls on soil carbon cycling and storage, and will be useful in both conceptual and empirical modeling of soil C. While this research is focused on mechanisms of carbon cycling and storage in surface soils, the results will be of interest to those aiding in on-farm decision making, as specific crop practices are assessed and connected to real gains in soil C. Changes/Problems:The primary challenge we are having with this project is the turnover of personnel. First, our start was delayed by COVID, and a student could not be brought on to being the experiment until 2021. The student we hired, Lucas Chamberlain, began working remotely in the spring of 2022, with the plan to move to Madison, WI in the summer in time to start field sampling. However, based on health needs of his family, decided to not move, but instead travel and stay for extended periods during the summer. By the end of the summer, he realized this was not a sustainable practice to complete a PhD and resigned his position. We immediatelybegan a search for a new graduate student and found one in December of 2021. This new student, Tanner Judd, will start in June of 2022. Two other PIs at other institutions have transitioned as well. Matt Liebman of Iowa State University has retired, and Yicaho Rui of the Rodale Institutehas taken a new job at Purdue University. We are working to address the lead PI voids left by these individuals. Overall, we still have complete capacity to conduct and finishthis research, but we will likely need an extension on the grant. What opportunities for training and professional development has the project provided?Unfortunately, with COVID and the transition from one graduate student to another, we have not yet had opportunities to provide professional development. How have the results been disseminated to communities of interest?To date, our research findings have only been communicated among the PI team at the University of Wisconsin-Madison, Rodale Institute, and Iowa State University. With the departure of the graduate student, a new field sampling plan and timeline was discussed. What do you plan to do during the next reporting period to accomplish the goals?The field research plan has been modified to accommodate and better train the incoming PhD student (Tanner Judd). In 2022, field research will be conducted in Wisconsin and in 2023, field research will be conducted in 2023. All field analysis will be conducted again in 2022, to provide annual replication of SOM fractionation, amino sugars, oxidative enzyme assays, CUE, and PMN. This will also allow for the training of the new graduate student on all techniques.

Impacts
What was accomplished under these goals? Following COVID-based delays, field sampling and laboratory analysis was conducted in 2021 at the Wisconsin integrated Cropping Systems Trial. Our approach was to sample in Wisconsin in 2021 and Pennsylvania and Iowa in 2022. Lucas Chamberlain, the PhD student on this project. Sampling: Briefly, eight 30cm soil cores were taken from each cropping system as close to corn roots as possible. The soil cores were split into two depths (0-15, 15-30cm) and homogenized to create one sample/ depth / system (n = 48). Sampling occurred twice across the 2021 growing season: May 26th and July 27th. (1 site x 6 cropping systems x 2 soil depths x 4 blocks x 2 time points = 96 total samples. Storage: After sieving (250mm), subsamples of each sample were placed into labelled 15mL and 50mL centrifuge tubes within Ziploc bags and stored at -80 C in the chest freezer outside of the Freedman Lab door. After analyses requiring microbial activity were completed (CUE, PMN and oxidative enzyme activity), all samples were air-driedat room temperature and placed in labelled Ziploc bags within a labelled. Analysis: Analysis was completed on SOM fractionation, carbon use efficiency, oxidative enzymes, and potentially mineralizable nitrogen for May samples. Analysis was completed on SOM fractionation, oxidative enzymes, and potentially mineralizable nitrogen on July samples. Samples have been properly stored to complete the carbon use efficiency analysis. However, the graduate student who conducted this analysis resigned from his position in September of 2021 and a new search for a graduate student began. A new PhD student, Tanner Judd was interviewed and hired in December of 2021, but did not start until June of 2022.

Publications

Progress 04/15/20 to 04/14/21

Outputs
Target Audience:The target audience for this effort period is the scientific community. Our starting work on this project only yet pertains to other scientists in terms of methodology and preliminary findings. Our future work will expand to applied audiences. Changes/Problems:Our main challenge for this experiment has been the turnover of our PhD student that will work on this project. Funding started in March of 2020, but due to COVD-19 restrictions and shutdowns, we were unable to recruita student to start until January of 2021. Thus, our immediate field work plan had been delayed by a year (from the 2020, 2021, and 2022 field seasons to the 2021, 2022, and 2023 field seasons). The student asked to work remotely (they still lived in Chicago) during the spring semester (which we approved), taking classes on-line and traveling up periodically to work on laboratory methods. The student began all initial field work at the Wisconsin site in the summer of 2021. However, it became clear that he was not willing to fully move to Wisconsin. Ultimately, he decided it was best to end is PhD pursuit in August of 2021 after finishing up the summer sampling. We were able to recruit another PhD student to begin in May of 2022 but will need to delay our field sampling at other sites to future years. We feel it is best that the new student be allowed to conduct his first year of research at a close location to ensure success. Travel to the Iowa and Pennsylvania sites for research will occur in 2023 and 2024 if allowed. What opportunities for training and professional development has the project provided?The project has trained 1 PhD student who opted to not continue after the first field season due to family health reasons and the COVD-19 pandemic. One undergraduate student research assistant has been supported by this project. While enrolled, the PhD student engaged with the soil science and soil microbiology community at UW-Madison, attended two grant writing workshops and a mock grant panel. The student was also awarded a small grant ($1,000) through the O.N. Allen Small Grants program at UW-Madison. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?A new PhD student will begin on May 15th, we anticipate that this PhD student will present data from this project at the Soil Science Society of America Annual Meeting in November 2022. We will continue to employ undergraduate research assistants as well. During the summer 2022 field season, we plant to determine the full suite of proposed data collection at the Wiscon site, collecting samples twice during the growing season. A manuscript will be prepared to disseminate findings across the 2021 and 2022 growing seasons.

Impacts
What was accomplished under these goals? A graduate student was hired and began training starting in January of 2021. The student began work toward objectives 1, 3, and 4. Soil sampling was performed at the Wisconsin site related to determining the quantity and quality of C inputs and microbial necromass. For objective 2, an optimized protocol for 18O carbon use efficiency and in situ stable isotope tracing is near completion (expected by summer of 2022). Soil samples from the Wisconsin site were sampled according to the proposal and data has been generated for the soil health metrics (potentially mineralizable nitrogen, permanganate oxidizible carbon, and enzyme activity). Fractionation of soil organic matter in to functionally distinct fractions (that is, particulate organic matter and mineral-associated organic matter) has been completed and the samples are awaiting total C and N analysis.

Publications