Progress report for GNC20-310
While agricultural soils account for a large portion of nitrogen pollution in the US, sustainable soil management practices present unmatched potential to slow global warming and counteract environmental externalities. Planting cover-crops is part of a set of recommended practices in conservation agriculture, a management approach that encourages farmers to decrease N losses by keeping living plants and plant cover in the soil during the off-season. Despite their promise in reducing sources of N loss through denitrification and leaching, the functional impact of cover-crops on microbially-driven soil N-cycling remains a mystery. At a time when cover-crops are gaining increased interest across the country, including in the Upper Midwest, leveraging below-ground interactions with microbes to tighten nutrient cycles presents the next big challenge for climate-friendly farming.
This project contributes to an existing SARE project aimed at engaging minority farmers in summer cover crop systems. Under the current project, optimal management of a variety of summer cover crop species is being investigated to maximize ecosystem services such as providing habitat for pollinators and plant-available mineral N. The objective of this proposal is to understand how summer cover-crop management impacts key soil microbial groups.
Cover crop management decisions such as species selection and termination date can influence microbial groups responsible for transforming soil N in ways that are not well-understood. Agriculturally-relevant sources of N loss that contribute to water pollution and greenhouse gas emissions are controlled by the key microbial processes of nitrification and denitrification. The objectives of this project are to 1) identify summer cover crop species and species mixtures that minimize soil N loss via impacts on key microbial communities, and 2) contribute to resources for underserved farmers to assess summer cover crop management trade-offs for ecosystem services. Farmer collaborators will assist in evaluating seven cover crop species mixture treatments, each at two different planting dates. Soil samples will be collected from on-farm trials at key timepoints to determine cover crop impacts on microbial populations. Information on how cover crop management interacts with microbial communities will be paired with qualitative farmer evaluation of the cover crop systems under study. This project will ultimately engage small-scale vegetable producers and minority farmers in making informed decisions on the unseen, below-ground impacts of planting cover-crops.
Objective 1. Identify summer cover crop species and species mixtures that minimize soil N loss through impacts on specific microbial communities. Learning outcome: 5 educators/researchers will learn about the impact of at least three summer cover-crop species on soil microbial groups that play a central role in soil N cycling. Action outcome: Educators will develop and distribute recommendations to 200 farmers for using summer-cover crops to functionally impact soil microbial communities.
Objective 2. Contribute to resources for immigrant and minority farmers to assess management trade-offs with summer cover crop ecosystem services. Learning outcome: 50 growers will learn to weigh impacts on soil microbial populations in their decision to adopt summer cover crop mixtures. Action outcome: Growers contribute to the preservation of plant-available soil N and reduce losses from leaching and denitrification by cultivating beneficial microbial communities through cover crop selection.
Soils were collected to a depth of 10 cm using ascetic technique to prevent microbial cross-contamination between treatments. Briefly, ten soil cores were collected from each plot, avoiding sampling from within 30 cm of the plot border. Before collecting soil cores, surface plant residue was removed. Cores from the same plot were pooled in sterile plastic bags. In between sampling different plots, a 70% ethanol solution was used to clean and sterilize the soil probe. Bulk soil samples were immediately placed in a cooler at kept at 4C until processing. All time-sensitive microbial enzyme assays were performed within one week of the sampling date. Within one week, a portion of the bulk soil sample was set aside and stored at -20C until DNA extractions could be performed.
Nitrification Potential (NP)
Nitrification potential (NP) refers to the maximum rate of activity among nitrifying organisms under optimal conditions. Determining the NP of a soil community can give us useful information as to how a specific management strategy or crop species influences the potential rate at which nitrogen is cycled through soil by microbes. High rates of NP during moments of rapid crop growth and N uptake are favorable; during other times, NP should be managed to reduce N loss to the environment. To measure NP in summer cover crops, we used a modified protocol from Kandeler et al. (2011) in Methods of Soil Enzymology (ed. Richard P. Dick).
In brief, a NP solution was made that consists of potassium phosphate and ammonium sulfate which provides nitrifying microbial communities with the substrate required for their growth and activity. The solution was calibrated to the pH of the native soil, so that the enzymatic potential obtained from the assay is more reflective of a true potential. Within one week of sampling, 2.5g of fresh soil was mixed with 25 ml of the NP solution and placed on an orbital shaker at 180 rpm for 36 hours. Over the course of the 36 hour incubation, the soil slurry is sampled four times: 2 hours, 12 hours, 24 hours, and 36 hours. Finally, nitrate (NO3- ) concentration was determined using colorimetric methods. NP was calculated as the rate of NO3- production over the course of the incubation period.
Denitrification Enzyme Activity (DEA)
DEA is a soil incubation assay that is similar to NP in that it measures the maximum potential activity of denitrifying organisms in soil under optimal conditions. For denitrifiers, 'optimal' conditions refer to anaerobic or anoxic conditions and an adequate amount of substrate: glucose as a carbon source and nitrate. High rates of DEA are seldom favorable, since the end products of the microbial denitrification pathway are gaseous forms of nitrogen (N2 and N2O) that are lost from the system. To measure the extent to which different summer cover crop plantings impact DEA rates, we used a modified protocol from Tiedje (1994).
A DEA solution was prepared using 1mM glucose and 1mM KNO3- and adjusted to the average pH of the soil samples for each location. Prior to running the assay, we prepped 9 ml gas sample vials by sealing them with airtight septa. Within one week of sampling, 2.5g of fresh soil and 10 ml of solution were added to 160ml serum vials. Both serum vials and sample vials were evacuated using a vacuum pump (-26.5 psi) and subsequently re-pressurizing to 15 psi with pure N2. This evacuation and pressurization cycle was repeated three times. On the final cycle, the pressure was brought down to 0.1 psi after flushing with N2. The start time for the incubation was counted as the time that C2H2 (10ml) was added to the sample vials. C2H2 serves to inhibit the final conversion of N2O to N2 in the denitrification pathway so that only a single product is obtained from the incubation. Samples were incubated for 90 min, and 10ml samples of the vial headspace were collected at 0, 45, and 90 min. Gas samples were stored in the prepped 9ml sample vials until analysis on a GC/MS, typically within one day of running the assay. DEA is reported as the rate of N2O produced during the course of the incubation.
Soil was stored at -20C prior to extracting DNA. Total genomic DNA was extracted from 0.25g fresh soil using Qiagen DNEasy Powersoil Pro kit. Extracted soil was again stored at -20C until amplicon sequencing for 16S (bacteria) and ITS2 (fungi).
Soil extractions for this experiment are ongoing. We expect to submit these samples for sequencing by March 2022.
Denitrification Enzyme Activity
On average across all timepoints, cover crop treatment did not influence denitrification enzyme activity (DEA) following spring-planted cover crop termination at either of the two study locations. We also analyzed differences in DEA among cover crop treatments within each sampling time point for each location. There were no differences among any of the treatments at any time point at BRF. In St Paul, the weed free control plot demonstrated lower DEA (p=0.05) than the pea/oat cover crop mixture at the first time point only (at cover crop termination; Fig 1).
DEA appeared to be more strongly influenced by seasonal rather than plant effects. Average DEA varied significantly between each sampling time point for both BRF and St Paul. At both locations, DEA was greatest at the second time point (30 days post termination) and then decreased at the third time point (60 days post termination). DEA was lower at the time of cover crop termination compared to the other time points at both locations. The second sampling time point occurred in August, when soil and moisture temperatures were at their highest levels of the season (Fig 1). Warm temperatures and high soil moisture are both factors that are known to increase microbial N cycling activity.
These results concur with our expectation that DEA would increase during the experiment as cover crop residues decomposed following termination. The initial time point at cover crop termination is a close representation of living cover effects on denitrifying microbial activity. BRF had lower soil organic matter then St Paul, which could account for non-significant treatment differences if carbon availability was a limiting factor for microbial growth. As expected in St Paul, the weed-free treatment exhibited lower DEA than the pea/oat mixture, which had the highest DEA. Notably, the legume-only treatment, crimson clover, did not have higher DEA than any of the other legume or non-legume treatments. These results suggest that crimson clover may be a promising summer cover crop to fill both nitrogen fertility and retention goals. Crimson clover contributes organic nitrogen to the soil via biological nitrogen fixation, but does not appear to stimulate denitrifying enzyme activity that would lead to N loss during residue decomposition.
Nitrification potential (NP) results were very similar to DEA results. The end product of the nitrification pathway, NO3-, is also the starting substrate for the denitrification pathway. Because there is facilitation of nitrogen products between nitrifying and denitrifying microorganisms, we expected NP and DEA results to be highly similar. At both BRF and StP, there was a strong and significant correlation between nitrification and denitrification enzyme activity across all timepoints (Fig 2).
As we observed with DEA, there was on average no difference in NP among cover crop treatments across all time points in either location. Within each time point at both locations, there was a trend for lower NP values in the weed free control treatment. This effect was marginally significant in StP for the third (p=0.08) time point and highly significant in BRF for the first time point when weed free plots had lower NP than all other treatments (p<0.001). This result was expected, since weed-free plots should have lower plant-derived nitrogen inputs compared to plots with living cover. In BRF, negative NP results obtained for the weed-free treatment (Fig 3) suggest potential immobilization, or uptake, of available soil nitrogen by microorganisms.
As cover crop and weed residues decomposed during the summer broccoli cash crop, we observed significant and linear increases in average NP for all treatments in BRF (Fig 3). In St Paul, NP did not increase linearly over the season. The highest rates of NP occurred 30 days after cover crop termination, while the lowest rates of NP occurred at the last time point, 60 days after termination. Even while NP and DEA are correlated due to the metabolic interdependence of the microorganisms involved, the differential effect of season on NP compared to DEA suggests that the nitrifying community responds differently to environmental factors. Nitrification is an aerobic process, so we would expect NP to exhibit a smaller response to increases in soil moisture as compared to DEA. Ongoing analysis involving our soil moisture and temperature probes will help to clarify specific environmental drivers on NP. In StP, the highest rates of NP (time point 2/ 60 days post-termination) coincided with an active period of broccoli growth. This implies that all cover crop treatments performed equally well in this location in supplying N during a critical period of cash crop growth.
Educational & Outreach Activities
Preliminary results from this study were presented at two professional meetings and one outreach event in 2021.
1. Summer research symposium at the University of Minnesota
In August 2021, we presented a poster at a University of Minnesota research forum that attracted educators and students from diverse disciplines. We reached an estimated number of 30 students and educators at this event. The topic of the presentation was "Microbial denitrification activity in summer cover crop mixtures".
2. ASA/CSSA/SSSA Annual Meeting
We presented complete results from the first field season of this study at the annual Agronomy Society of America/Crop Science Society of America/Soil Science Society of America meeting in November 2021 in Salt Lake City, Utah. The title of our poster presentation was "Maximizing summer cover crop conservation benefits for improved vegetable production". We engaged with an estimated number of 50 scientists, students, educators, and extensionists at this event.
3. Minnesota State Fair Outreach and Education Booth
In August 2021, we facilitated a 4-hour long outreach and extension booth in the Horticulture Building at the Minnesota State fair. We interacted with an estimated number of 50 members of the general public, which included students, homeowners, and farmers. At this event, we conducted a hands-on experiment to demonstrate the role of microorganisms in providing soil nutrients and contributing to health soil structure. We also displayed different cover crop options, and provided advice on when/why to apply microbial inoculants.
Upcoming outreach events include hands-on farmer workshops on microbial contributions to soil fertility at the Great Lakes Indigenous Farming Conference (March 2022), Big River Farms (2022), and the Emerging Farmers Conference (November 2022). We are also presenting first-year findings at the MOSES Organic Farming Conference in February 2022, which will provide an excellent opportunity to engage directly with growers in the Upper Midwest. Pending public health guidance, we hope to hold at least one field day during the 2022 growing season.
At one or more of these upcoming events, we plan to distribute infographics explaining the role of microorganisms in cycling soil nutrients. In line with our grant objectives, we also plan to disseminate a survey at one or more of these planned events in 2022 to understand how farmers weigh fertility-enhancing services from microbes in comparison to other benefits from summer cover crops such as attracting beneficial pollinators.
Spring-planted cover crops provide organic vegetable farmers with an opportunity to introduce a diversity of ecosystem services into their summer cash crop rotations. In the short growing season of the upper Midwest, identifying optimal cover crop mixtures that maximize services such as soil fertility within a limited window of time remains a challenge. In particular, greater knowledge of how cover crops interact with soil microbiota will help to inform growers with a balanced perspective of potential nitrogen fertility and nitrogen retention services. Here, we assessed how cover crop residue decomposition affects nitrogen cycling microbial activity across three treatments that were identified by our farmer collaborators: a non-legume (buckwheat), a legume only (crimson clover), and a grass/legume mixture (pea/oat). Our preliminary findings suggest that crimson clover may offer the best balance of N retention and provision services because it did not stimulate potential N loss due to denitrifying soil activity during the warmest and wettest period of residue decomposition in mid-August.
We are in the first phase of this two-year study. This grant has greatly helped to improve our awareness of how summer cover crop decomposition processes impact soil microbial activity. We were surprised to find few differences between legume and non-legume cover crop treatments. These preliminary results could suggest that legume cover crops provide a longer-term, stable source of organic nitrogen for cash crop growth without enriching microbes that would rapidly metabolize nitrogen inputs, leading to its loss. In particular, crimson clover appears promising because it did not stimulate nitrifying or denitrifying activity relative to other treatments, including controls. Crimson clover has also performed well in recruiting beneficial insects, which is an emerging finding from the larger SARE grant to which this student fellowship contributes. This work allows us to holistically consider multiple ecosystem services from summer cover crops and provide recommendations to growers in year two of the study.