ABSTRACT Title of Dissertation: DEEP SOIL NITROGEN CAPTURE AND RECYCLING BY EARLY-PLANTED, DEEP- ROOTED COVER CROPS Sarah M. Hirsh, Doctor of Philosophy, 2018 Dissertation directed by: Dr. Ray R. Weil, Department of Environmental Science and Technology The overall purpose of this study was to improve the efficiency of nitrogen (N) cycling in Mid-Atlantic cropping systems through the use of cover crops. Our focus was on describing soil inorganic N pools (0-210 cm deep) and investigating the potential for cover crops to scavenge and recycle deep soil N. Few agronomic studies consider soil properties and processes deeper than the upper 20 to 30 cm, as the majority of roots, amendments, and practices such as fertilizer application or tillage occur on the soil surface or in the topsoil. We 1) assessed amounts of deep soil N on 29 farms in the Mid-Atlantic region, 2) used 15N tracer to investigate the capacity of various cover crops with early- or late-planting dates to capture and recycle deep soil N, and 3) investigated early-planted cover crop systems on 19 farm trials to assess their performance on farms with various soils with diverse management practices. We found that on average 253 kg N ha-1 of inorganic N remained in the soil following summer crops, 55% from 90-210 cm deep. Soil following soybean had the same amount or more of inorganic N than soil following corn throughout the soil profile. Using 15N isotopic tracer, we determined that radish, rye, and radish/rye mixes with and without crimson clover all could capture N from deep soil (60+ cm), but in order for cover crops to capture agronomically meaningful amounts of nitrate-nitrogen (NO3-N) from deep soil, they had to be planted by early-September. Cover crop trials on 19 farms indicated that, while variable site-by-site, early-planted cover crops tended to accumulate substantial N in the fall and reduce residual soil NO3-N levels substantially in the fall and spring. Cover crops also impacted subsequent corn growth and yield, with winter cereal tending to cause lower yields or increased corn N fertilizer needs compared to a no cover crop control, and forage radish sometimes leading to higher yields compared to the control. Overall, cover crops are effective at scavenging deep soil N in the fall, before winter leaching occurs, and under certain conditions, can release N for subsequent crops. DEEP SOIL NITROGEN CAPTURE AND RECYCLING BY EARLY- PLANTED, DEEP-ROOTED COVER CROPS by Sarah M. Hirsh Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2018 Advisory Committee: Dr. Ray R. Weil, Chair Dr. Robert Kratochvil Dr. John Meisinger Dr. Steven Mirsky Dr. Katherine Tully © Copyright by Sarah M. Hirsh 2018 Acknowledgements I would like to thank my advisor, Dr. Ray Weil, and committee members Dr. Robert Kratochvil, Dr. John Meisinger, Dr. Steven Mirsky, and Dr. Katherine Tully. I would like to thank Extension collaborators from University of Maryland: Stan Fultz, Jim Lewis, and Matt Morris, and collaborators from Pennsylvania State University: Dr. Sjoerd Duiker, Jeff Graybill, and Kelly Nichols. I would like to thank our student workers and interns, and all of the farmers who collaborated with us and graciously offered their land, resources, and time. This project was supported by: Northeast SARE and Maryland Soybean Board. ii Table of Contents Acknowledgements ...................................................................................................ii Table of Contents .................................................................................................... iii List of Tables............................................................................................................ iv List of Figures ........................................................................................................viii Chapter 1: Literature review ...................................................................................... 1 Chapter 2: Cropland soil profiles in the Mid-Atlantic contain large pools of residual inorganic N .............................................................................................................. 27 Abstract ............................................................................................................... 27 Introduction ......................................................................................................... 28 Materials and Methods ......................................................................................... 31 Results ................................................................................................................. 38 Discussion ........................................................................................................... 40 Appendix 1. Transect site soil characteristics ....................................................... 58 Appendix 2. Detailed procedures for soil coring.................................................. 72 Appendix 3. Soil nitrate and ammonium calculations ........................................... 75 Appendix 4. Soil bulk density values .................................................................. 76 Chapter 3: Cover crop species and planting date affect deep soil nitrate capture ....... 79 Abstract ............................................................................................................... 79 Introduction ......................................................................................................... 80 Materials and methods ......................................................................................... 86 Results ............................................................................................................... 105 Discussion ......................................................................................................... 110 Appendix 5. Detailed methods for estimation of rye biomass ............................. 132 Appendix 6. 15N percent recovery data ............................................................... 133 Chapter 4: Cover crop systems influence on deep soil N dynamics and the following corn crop: on-farm investigations........................................................................... 136 Abstract ............................................................................................................. 136 Introduction ....................................................................................................... 137 Materials and Methods ....................................................................................... 140 Results ............................................................................................................... 150 Discussion ......................................................................................................... 157 Appendix 7. Supplemental soil characteristics of study sites .............................. 191 Appendix 8. Cover crop sampling details ........................................................... 197 Appendix 9. List of weather stations .................................................................. 198 Appendix 10. Bulk density of soil cores ............................................................. 199 Appendix 11. Corn yield equations .................................................................... 201 Chapter 5: Policy implications and Conclusion ..................................................... 202 Bibliography .......................................................................................................... 206 iii List of Tables Table 1. Site descriptions for soil core transect sites, indicating crop, manure, and tillage history, and soil descriptions. Physiographic regions were determined according to Polsky, et al. (2000). Soil series and phase were determined from Web Soil Survey (WSS) data from USDA NRCS (https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm); soil sample texture was compared to the official soil series descriptions from USDA NRCS (https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/home/?cid=nrcs142p2_05358 7) to ensure soil samples correlated to the mapping units. Parent material classification based on soil series description and verified with observed soil characteristics (e.g., pH and texture). ....................................................................... 45 Table 2. Soil NO3-N, NH4-N, and mineral N (NO3-N + NH4-N) (kg N ha -1) for 0-30 cm, 30-90 cm, 90-150 cm, 150-210 cm, and 0-210 cm, and the percent of total mineral N found in each soil depth increment. Values are average of all sites (N=29), Coastal plain sediments sites (N=14), Calcareous rocks sites (N=6), and Acidic rocks sites (N=9). Within a depth increment, values followed by the same lower case letter do not differ significantly......................................................................................... 50 Table 3. Twenty-nine farm mean, standard deviation (SD), and range values of soil NO3-N (kg N ha -1), NH4-N (kg N ha -1), and NO3-N percent of the total mineral N (NO3-N + NH4-N), pH, percent sand, clay, and silt, percent total C, percent total N, and C/N ratio. Soil divided into increments of 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm. The percent total N and C/N ratio calculated for 0-30 cm increment only, due to many below detection limit (BDL) N levels in deeper layers. ............... 51 Table 4. Correlations coefficient (r) and significance (p-value) for correlations between soil NO3-N (kg N ha -1), NH4-N (kg N ha -1), and NO3-N percent of the total mineral N (NO3-N + NH4-N) with soil percent sand, clay, silt, total C, total N, C/N ratio, and pH. Data for 29 farms analyzed by profile increments of 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm. The percent total N and C/N ratio correlated for 0- 30 cm increment only, due to N levels below detection limit. ................................... 52 Table 5. Study site soil pH, percent sand, percent clay, soil texture, percent C, and percent N for each 15 or 30 cm soil depth increment (0-210 cm), and percent soil organic matter (SOM), P, K, Mg, Ca, and S (mg kg-1) for the upper 30 cm of soil from each site. Each record is the average of two to three composited soil cores from two areas in the field. Data from Dorchester IB 180-210 cm, Lancaster IB 180-210 cm, and St Marys I 195-210 cm is from a single point of a field. Values not determined indicated as nd. Values below detection limit indicated as BDL............. 58 Table 6. Bulk density (BD) mean (g cm-3), standard deviation (SD) (g cm-3), first (Q1) and third (Q3) interquartile range (25th to 75th percentiles) and number of outliers above fences for all farms in which soil cores were taken. The fence is defined as 1.5 x Interquartile range (25th to 75th percentiles). ................................... 77 iv Table 7. Experiment #1, soil pH, percent sand, percent clay, percent C, percent N, NH4-N (kg N ha -1), and NO3-N (kg N ha -1) for each 20 cm soil depth increment (0- 200 cm) and P, K, Mg, Ca, and S (mg kg-1) for 0-30 cm soil. Reported pH, sand, clay, C, N, P, K, Mg, Ca, and S values are the average from three soil cores, one per block. Reported NO3-N and NH4-N values are the average from six soil cores, two per block. .................................................................................................................... 115 Table 8. Experiment #2, soil pH, percent sand, percent clay, percent C, percent N, NH -N (kg N ha-14 ), and NO3-N (kg N ha -1) for each 15 cm soil depth increment (0- 210 cm) and P, K, Mg, Ca, and S (mg kg-1) for top three 15 cm soil depth increments (0-45 cm). Reported pH, sand, clay, C, N, P, K, Mg, Ca, and S values are the average from six cores (two cores 10 cm apart composited, taken in each of three blocks). Reported NO3-N and NH4-N values are the average from 15 cores (five cores in each of three blocks). ..................................................................................................... 116 Table 9. Experiment #2, experimental treatment combinations. Experimental factors that defined the treatments included cover crop, cover crop planting date, and 15N burial depth. The cover corps indicated in white were only planted early, not late. The cover crops indicated in grey were planted early and late. ...................................... 118 Table 10. Experiment #2, soil samples taken (and depths analyzed in parentheses) per block. The number of asterisks indicate the number of composite cores per sample. Two cores from the same distance from the tracer (dist 15N) were from two different burial points within a plot; two cores from different dist 15N were from one burial point within the plot. .............................................................................................. 119 Table 11. Experiment #1, analysis of variance (ANOVA) tests of fixed effects for log10 15N percent recovery .................................................................................... 120 Table 12. Experiment #2, percent of the six replications within a given treatment with cover crop tissue type at% 15N significantly (p < 0.01) above background (no 15N application) level. .................................................................................................. 121 Table 13. Experiment #2, 15N percent recovery for fall and spring rye. The p-values indicated differences between fall and spring log10 15N percent recovery. ............. 122 Table 14. Experiment #2, percent of the six replications within a given treatment with V5 corn or corn grain at% 15N significantly (p < 0.01) above background (no 15N application) level. .................................................................................................. 123 Table 15. Samples taken to estimate rye biomass, the number of samples (N) taken per treatment, regression equations relating rye patchiness, height, and/or percent cover to biomass, and adjusted R2 for each regression equation ............................. 132 Table 16. Percent recovery of 15N for December sampled radish, rye, two-way mix (2mix), and three-way mix (3mix) cover crops, showing the number of observations per reported value (N), mean, standard deviation (SD), minimum value (Min) and maximum value (Max) .......................................................................................... 133 Table 17. Percent recovery of 15N for April sampled two-way mix (2mix), three-way mix (3mix), and rye cover crops, showing the number of observations per reported value (N), mean, standard deviation (SD), minimum value (Min) and maximum value (max) ..................................................................................................................... 135 v Table 18. Site histories of cover crop studies. ........................................................ 165 Table 19. Cover crop treatments, planting date, management details, and sampling timing of cover crop studies. .................................................................................. 167 Table 20. Corn yield and N response trials. Cover crop termination date, corn planting date, N fertilizer type, date applied and rates, corn herbicide information, samples taken and sampling method.................................................................................... 171 Table 21. Soil NO3-N and NH4-N (kg ha -1) of radish, winter cereal (cereal), mixed species (mix), and control cover crop treatments for six farms for late-fall sampling and for 11 farms for spring sampling. Different letters indicate statistically significant differences between cover crop treatments per depth increment. Farms sampled in late-fall include Dorchester IB, Frederick IV, Huntington IA, Lancaster IA, Lancaster II, and Lancaster V. Dorchester IB cores only to 180 cm deep, Frederick IV did not have soil core samples from mix treatment. Farms sampled in spring include Dorchester IB, Frederick I, Frederick III, Howard IB, Huntington IA, Lancaster IA, Lancaster IB, Lancaster II, Lancaster III, Lancaster V, and Kent II. Dorchester IB and Kent II soil cores were to only 180 cm deep. Lancaster V did not have soil core samples from mix treatment. .................................................................................. 173 Table 22. Fall and spring sum of NO3-N (kg ha -1) and NH4-N (kg ha -1) from 0-90 cm and from 0-210 cm deep for 14 farms, and cover crop biomass (kg ha-1), N content (kg N ha-1), and C/N ratio for 19 farms. Cover crop treatment values for a response variable, within the same season and farm, followed by the different letters are significantly different (p <0.05); * indicates significantly different (p < 0.1). ......... 175 Table 23. Cover crop biomass (kg ha-1), N content (kg N ha-1), and C/N ratio for fall cover crop growth (late-fall sampling), fall and spring cover crop growth (spring sampling), and cover crop growth prior to termination (late-fall radish, prior to winter-kill, and spring winter cereal, prior to herbicide termination. Different letters indicate statistically significant differences between cover crop treatments. ........... 180 Table 24. Correlations among cover crop biomass and growing degree days (GDD), precipitation (prec.) total from cover crop planting date to sampling date, topsoil (0- 30 cm) NO3-N and NH4-N (kg N ha -1), topsoil percent sand/clay/silt. Table showing number of replicates (N), correlation coefficient (r), and p-value of correlation...... 181 Table 25. June PSNT soil sample NO3-N and NH4-N concentrations (mg N kg -1 soil), and corn plant biomass per corn plant (g plant-1) and N in biomass per corn plant (g N plant-1) following cover crop treatments. N indicates the number of replicates per cover crop treatment. Cover crop treatment values for a response variable, within the same farm, followed by the different letters are significantly different (p < 0.05). .. 182 Table 26. Percent of maximum corn yield following cover crop treatments for farmers’ standard fertilizer application rate (standard) or no fertilizer application. Cover crop treatment values for percent of maximum corn yield, within the same fertilizer N level, followed by different letters are significantly different (p < 0.05). .............................................................................................................................. 183 Table 27. Study site soil pH, percent sand, percent clay, percent C, and percent N for each 15 or 30 cm soil depth increment (0-210 cm), and percent soil organic matter vi (SOM), P, K, Mg, Ca, and S (mg kg-1) for the upper 30 cm of soil from each farm site. Each record is the average of two to three composited soil cores from two areas in the field. Data from Dorchester IB 180-210 cm and Lancaster IB 180-210 cm is from a single point of a field. Soil samples were not taken from Franklin IIA or Huntington IB; however these sites were within 100 meters of Franklin IIB and Huntington IA, respectively. Values not determined indicated as nd. Values below detection limit indicated as BDL. ........................................................................... 191 Table 28. Fall and spring cover biomass number and size of quadrats collected from each plot. ............................................................................................................... 197 Table 29. Distance from weather station to farm for precipitation and temperature measurements. ....................................................................................................... 198 Table 30. Bulk density (BD) mean (g cm-3), standard deviation (SD) (g cm-3), first (Q1) and third (Q3) interquartile range (25th to 75th percentiles) and number of outliers above fences for all farms in which soil cores were taken. The fence is defined as 1.5 x Interquartile range (25th to 75th percentiles). ................................. 199 vii List of Figures Figure 1. Locations in Maryland and Pennsylvania of the 29 crop fields in which a transect of 0-210 cm deep soil cores were taken. ...................................................... 53 Figure 2. Deep soil core placement scheme for (a) 2014 showing placement of all five sets of cores per field, and (b) 2015 showing positions for one of the four sets of cores per field. .................................................................................................................. 54 Figure 3. Twenty-nine farm 0-210 cm NO3-N (kg N ha -1) and NH4-N (kg N ha -1). Error bars show standard error (SE) of mean. Sites Dorchester IB and Lancaster IB total is for 0-180 cm only. ........................................................................................ 55 Figure 4. Amount of NO3-N and NH4-N (kg N soil layer -1 ha-1) and NO3-N percent of the total mineral N (NO3-N + NH4-N) of each 30 cm depth increment for sites with Coastal Plain sediments, Acidic rocks, and Calcareous rocks parent materials. Different lowercase letters indicate significant differences among depths within each parent material group. Different uppercase letters indicate significant differences among parent material groups within each depth. ..................................................... 56 Figure 5. NO3-N and NH4-N (kg N soil layer -1 ha-1) in four pairs of adjacent corn and soybean fields. The symbols **, *, †, ns indicate p < 0.01, 0.05, 0.1, and not significant. ............................................................................................................... 57 Figure 6. Veihmeyer probe, hammer, jack and lever system, and PVC troughs used for taking deep soil cores. ........................................................................................ 74 Figure 7. Experiment #1, temperature (°C) and precipitation (mm) from 1 Sep 2014 to 30 Nov 2014. ......................................................................................................... 124 Figure 8. Experiment #1, split-split plot experimental design and treatments, showing cover crop planting date as the main plot factor, cover crop as the split-plot factor, and 15N burial depth as the split-split plot factor. .................................................... 125 Figure 9. Experiment #2, temperature (°C) and precipitation (mm) from 3 Sep 2015 to 7 Oct 2016. ............................................................................................................ 126 Figure 10. Experiment #2, horizontal spatial arrangement of 15N burial holes (red dots) and areas around burial points in which biomass was sampled (green circles). .............................................................................................................................. 127 Figure 11. Experiment #1, soil at% 15N in soil cores taken in December 2014 (site one) and May 2015 (sites one and two) from early-planted rye plots in which 15N was buried at 100 cm. The at% 15N values are the average of two blocks in December and three blocks in May. .............................................................................................. 128 Figure 12. Experiment #2, December 15N percent recovery from each 15N burial depth for early- and late-planted cover crops (across all cover crops). The 15N burial depth values for log10 15N percent recovery within the same cover crop planting date treatment followed by the different letters are significantly different (p < 0.05). .... 129 Figure 13. Experiment #2, soil at% 15N in soil cores taken in February 2016 (site three) or April 2016 (site four), June 2016 (sites three and four), and October 2016 (sites three and four) from the no cover crop control plots in which 15N was buried at 60 cm. The at% 15N values are the average of three blocks. ................................... 130 viii Figure 14. Experiment #2, soil at% 15N in soil cores taken in February 2016 (site three) or April 2016 (site four) from the no cover crop control plots in which 15N was buried at 120 cm. The at% 15N values are the average of three blocks. ................... 131 Figure 15. Cover crop N uptake (CC N; green bars) and soil NO -13-N (kg ha ) from 0- 90 cm (brown bars) and 90-210 cm (orange bars) for 13 farms with biomass and soil samples collected. Belowground N (soil) indicated with negative values. Standard error bars show. ..................................................................................................... 184 Figure 16. Amount of NO3-N (kg ha -1) in 0-210 cm soil profile for seven farms at fall sampling and 10 farms at spring sampling. 1Spring samples from 120-150 cm and 150-180 cm depths are the average values from 120-180 cm.................................. 186 Figure 17. Relationship between fraction of maximum corn yield (no N applied on corn) and pre-sidedress test nitrate concentrations for cover crop treatments. Data from Howard IB, Franklin IIB, and Lancaster IB. .................................................. 188 Figure 18. Corn biomass at V5 growth stage for cover crop treatments. Corn biomass values with different letters are significantly different (p < 0.05). Data from Dorchester IB, Frederick IV, Howard IB, Franklin IIB, Kent II and Lancaster IB. *Differences between radish and control at p < 0.0561. ......................................... 189 Figure 19. Corn grain yield associated with various N fertilizer rates and preceding cover crop treatments. Error bars show standard error of mean. ............................. 190 ix Chapter 1: Literature review The following review will illustrate the importance of deep soil (1 m or more in depth) nitrogen (N) for improving nutrient use efficiency in agriculture and reducing N leaching loss to the environment. We will discuss the expected amounts of residual N in agricultural soil. We will next introduce cover crops, and discuss cover crop biomass and N accumulation, cover crop rooting dynamics, cover crop effects on residual soil N, and cover crop decomposition, nutrient release, and yield of subsequent cash crops. Nitrogen dynamics in cropland Nitrogen use efficiency in agriculture is a key issue for both maximizing farm profits and minimizing environmental impacts of farming. The N cycle is complicated, involving many inputs, outputs, and transformations. It is important to keep in mind that approximately 95-99% of N in the soil is in organic forms that are unavailable for plant uptake, and that plant available N, primarily the inorganic forms of nitrate (NO3-N) and ammonium (NH4-N), is released during microbial decomposition, which is dependent on various environmental and site-specific factors (Dahnke and Johnson, 1990; Weil and Brady, 2017). For example, the soil pH can enhance nitrification rates such that when soil pH increases 4.7 to 6.5, nitrification rates increase three to five times (Dancer, et al., 1973). Inorganic N forms can be leached, fixed on clay particles, and lost as gasses (N2, N2O, NO, NH3). Van Meter, et al. (2016) found evidence from multiple long-term studies that total N (organic and inorganic) can accumulate over decades in fertilized row crop agriculture soils in the Midwest USA. In Iowa, assuming a linear rate of change, total N was estimated to have accumulated over 50 years at a rate of 31 kg ha-1 yr-1 in soils to 1 1 m depth. Total N increased in the 25-50 cm depth by 22%, in the 50-75 cm depth by 20%, and in the 75-100 cm depth by 14%. In Illinois, assuming a linear rate of change, total N was estimated to have accumulated over 45 years at a rate of 70 kg ha-1 yr-1 in the 0-100 cm deep soil. Total N increased in the 20-50 cm depth by 27% and in the 50-100 cm depth by 66%. Furthermore, an analysis of 2069 NCSS (National Cooperative Soil Survey) soil samples from the six sub-basins of the Mississippi River Basin found total N was estimated to have accumulated over 30 years at a rate of 55 kg ha-1 yr-1 in the 0-100 cm deep soil. Strickland, et al. (2015) found on loamy sand and sandy loam soils in Georgia, that incorporating conservation practices including a cereal rye (Secale cereale L.) and winter pea (Lathyrus hirsutus L.) cover crops, increased the amount of total N in soil (0-65 cm) on average 2000 kg ha-1 over three years. Regardless of fertilizer amounts, substantial residual soil nitrate-N (NO3-N) and ammonium-N (NH4-N) is found in the soil at the end of the crop growing season. On farms in central and southeastern Pennsylvania, in the fall following corn growth, when N was applied at economic optimum rates, there was on average 74 kg NO3-N ha -1 and 94 kg NO3-N ha -1 in the 0-120 cm deep soil for non-manured and manured sites, respectively (Roth and Fox, 1990). Under various wheat (Triticum aestivum L.) systems in a loam soil in Saskatchewan, Campbell, et al. (2006) found fall NO3-N following wheat harvest in soil (0-2.4 m deep) was between 100 and 150 kg ha-1, despite receiving fertilizer amounts based on soil tests. On a Willamette loam soil in Oregon, following an unfertilized winter wheat crop (in September), 0-120 cm soil had 44 kg NO3-N ha -1 and 32 kg NH4-N ha -1, and following an unfertilized broccoli (Brassica oleracea var. italica) crop, 0-120 cm soil had 34 kg NO3-N ha -1 and 26 kg NH -14-N ha . With the recommended 2 rate of fertilizer, following winter wheat, 0-120 cm soil had 64 kg NO3-N and 37 kg NH4- N ha-1, and following broccoli, 0-120 cm soil had 180 kg NO3-N and 375 kg NH4-N ha -1 (Brandi-Dohrn, et al., 1997). In Minnesota, on a Webster clay loam soil, which had not been fertilized with inorganic N or manure for > 10 years, following an unfertilized corn crop, there was 71 to 91 kg N ha-1 residual NO3-N in the 0-3 m deep soil in the fall (Gast, et al., 1978). In the Northeastern USA, manured fields generally have higher residual N in 0-120+ cm soil and N loss due to leaching as opposed to non-manured fields (Angle, et al., 1993; Jokela, 1992; Roth and Fox, 1990; Weil, et al., 1990). In irrigated sandy soils in Maryland, Weil, et al. (1990) found that spring applications of manure to corn fields resulted in increased groundwater NO3-N levels within one year of the manure application. The proportion of NO3-N to NH4-N can vary widely. It is not uncommon, especially on manured soils, for NH4-N concentrations to be as high or even higher than NO3-N concentrations (Brandi-Dohrn, et al., 1997; Eghball, et al., 2004; Kristensen and Thorup-Kristensen, 2004b; Lacey and Armstrong, 2015; Sainju, et al., 2007). Greater NH4-N levels could be attributed to ammonification exceeding nitrification due to higher soil water content or due to NH4-N retention on clay particle cation exchange sites in the subsoil (Sainju, et al., 2007). Previous studies have found NO3-N levels to be more dynamic than NH4-N levels. Kristensen and Thorup-Kristensen (2004b) found that October residual NO3-N (0- 2.5 m) varied between crop species, with sweet corn (Zea mays L. Saccharata Koern.) > carrot (Daucus carota L.) > white cabbage (Brassica oleracea L. convar. Capitata), whereas residual NH4-N did not vary between the different species. From soil cores taken 3 in various barley (Hordeum vulgare L.), fescue (Festuca L.), and alfalfa (Medicago sativa L.) cropping systems (samples 1 m deep, six to nine times per year), Bergstrom (1986) found NH4-N did not vary much between treatments, staying between 11 and 13 kg N ha-1, whereas NO3-N ranged between 23 and 68 kg N ha -1. On a silt loam soil and a loamy sand soil in Wisconsin, Bundy, et al. (1993) found that spring soil NO3-N (0-90 cm) was higher following soybean in a corn/soybean rotation than following corn in a no- fertilizer continuous corn rotation, but there was no consistent effect of corn/soybean sequence on NH4-N levels. Soil inorganic N might be expected to increase following corn versus soybean cash crops since corn receives N fertilization, while soybean, a legume, does not usually receive N fertilization. However, previous studies found that corn did not have higher residual soil NO3-N levels than soybean following crop harvest (Jaynes, et al., 2001; Pantoja, et al., 2016; Rembon and MacKenzie, 1997). In Nebraska on a Sharpsburg silty clay loam soil, Kessavalou and Walters (1999) found that May soil residual NO3-N (0- 150 cm) was lower in a continuous corn system than following corn in the corn/soybean rotation system, even though it was fertilized more often (every year) and had 25% less N removed in corn yield than the corn in the corn/soybean rotation. Mineral N may be higher following soybean than corn because the soil in a soybean crop is a high N environment with low C/N residues and high N root exudates. Microbial N immobilization, which would remove NO3-N and NH4-N from the solution, would be expected to be much lower under soybean than under corn. Green and Blackmer (1995) found that rates of N mineralization did not differ in soils having soybean residue from soils having corn residue. However, they suggested that there is higher N immobilization 4 following corn, due to the larger amount of corn residue than soybean residue, which allows N to be more available following soybean (Green and Blackmer, 1995). Nitrate leaching and environmental concerns The combination of cropping systems, the humid climate and weather patterns, and soil characteristics in the Mid-Atlantic USA make agricultural systems in this region prone to NO3 leaching. A common cropping system in the Mid-Atlantic region is a corn (Zea mays L.) to soybean (Glycine max (L.) Merr.) rotation. In this rotation, from September to May there is no crop actively taking up N from the soil (Meisinger, et al., 1991), and little evapotranspiration (ET), but levels of precipitation remain equivalent to summer (Meisinger and Delgado, 2002). Leaching as NO3 can be the main pathway for loss of N from farmland in the Mid-Atlantic, when there is little vegetation growing on cropland and precipitation is greater than ET (Meisinger, et al., 1991; Shipley, et al., 1992). The location of the residual N is of particular importance, as the deeper N is in the soil profile, the more likely it is to be lost through leaching or become inaccessible for following crop roots (Thorup-Kristensen, 1994). Nitrate that is leaching through the soil from August through May will likely be out of reach for the subsequent corn crop. Nitrate leaching poses environmental risks, as NO3 can enter groundwater and bodies of water, such as the Chesapeake Bay. According to the Chesapeake Bay Model, agriculture is responsible for approximately 43% of the N getting into the bay—17% from chemical fertilizer, 19% from manure, and 7% from air deposition of ammonia from livestock (e.g., emissions from poultry houses and dairies) and agricultural soil emissions (Environmental Protection Agency, 2010). Furthermore, in the Chesapeake Bay watershed, approximately 50% of the N load in streams was transported through 5 groundwater (Phillips and Lindsey, 2013). Excessive N and phosphorus (P) loading in the Chesapeake Bay and its tributaries have caused eutrophication—leading to harmful algal blooms, decreased water clarity, and decreased submerged aquatic vegetation—and periods of hypoxia (dissolved-oxygen concentration < 1.0 mg L-1), stressing and killing aquatic organisms (e.g., shellfish; Ator and Denver, 2015; Phillips and Caughron, 2014). Maryland law requires that farmers grossing > $2500 year-1 follow nutrient management plans, which indicate the nutrient sources (e.g., fertilizer, manure) that can be added to crops. The Maryland Department of Agriculture Nutrient Management Program intends to protect “water quality in the Chesapeake Bay and its tributaries by ensuring that farmers and urban land managers apply fertilizers, animal manure and other nutrient sources in an effective and environmentally sound manner” (Maryland Department of Agriculture, 2014). The Chesapeake Bay Watershed Implementation Plans (WIPs) indicate how the Bay jurisdiction states (Delaware, Maryland, New York, Pennsylvania, Virginia, West Virginia, and the District of Columbia) will meet Total Maximum Daily Load (TMDL) goals of reducing N, P and sediment inputs into the Bay watershed. The EPA (Environmental Protection Agency) Interim Evaluation of Maryland’s 2016-2017 milestones reports that the Agriculture sector in Maryland was not on-track to reach its 2017 N target, which is a 60% reduction of the 2009 N loads into the Bay to achieve water quality standards. Specifically, 10 monitoring station sites indicated decreasing N load trends, two sites indicated no significant trend, and six sites indicated increasing N load trends (Environmental Protection Agency, 2017). Therefore, even with statewide, mandated efforts, N leaching continues to be a concern in Maryland. 6 It is important to note it is desirable to have plentiful N in the soil at certain times of year and in certain soil layers. Agricultural production is dependent on large pools of available N in the soil. However, how deep the N is located is key. Thorup-Kristensen (2006a) noted that the downward movement of N is not a loss process, but rather the loss occurs if the N leaches beyond the rooting zone of crops. In other words, N can move down through the soil profile, without actually being lost from the system, if it remains within the rooting zone of crops. Therefore, the loss of N from leaching is largely influenced by the amount of precipitation and infiltration (Thorup-Kristensen, et al., 2003). Cover cropping to improve nitrogen use efficiency Plants grown in the fall, following harvest of the cash crop, are called cover crops, catch crops, or green manures (Thorup-Kristensen, et al., 2003). Some cover crop species have the potential to quickly grow deep roots, and could serve as a “catch crop” to capture NO3 in the fall months before it leaches out of reach, and potentially release N in the spring months to be used by the following cash crop (Dabney, et al., 2010; Meisinger, et al., 1990; Meisinger, et al., 1991). Cover crops can serve to reduce NO3 concentration in aquifers used for drinking water and to decrease NO3 concentrations in surface waters, lessening the risk of eutrophication and associated negative environmental effects (Thorup-Kristensen, et al., 2003). Cover crops can be fit within the framework of the existing crop system to scavenge and accumulate N in their tissue, and then through their decomposition, to supply N for subsequent crops. Cover crops can improve the N use efficiency of a corn cropping system. Corn tends to scavenge the majority of its N from upper soil layers, especially after high 7 fertilization applications (Gass, et al., 1971; Ju, et al., 2007). On a loam soil, corn rooting depth was found to be < 0.8 m at V9 (nine leaf collar) stage and reached a maximum root depth of 1.2 m at silking stage (Zhou, et al., 2008). However, while corn roots did not reach depths > 1.2 m, subsequent winter wheat could use soil NO3 up to 2 m deep (Zhou, et al., 2008). Using 15N tracer, Huang, et al. (1996) found that corn only removed 1 kg NO3-N ha -1 from 120 cm deep soil, whereas switchgrass (Panicum virgatum L.) removed 20 kg NO3-N ha -1. Three main functional groups of cover crops that are grown include winter cereal grasses, legumes, and brassicas (Dabney, et al., 2010). In the current study, the three cover crop species studied in the 15N experiment, and in most of the on-farm trials, were forage radish (Raphanus sativus L.), cereal rye, and crimson clover (Trifolium incarnatum L.); however other winter cereal species were studied rather than cereal rye on some on-farm trials. Tribouillois, et al. (2015) found that the crop growth rate and N acquisition rate for forage radish was greater than crimson clover, which was greater than cereal rye. In Maryland, Dean and Weil (2009) found that early-planted forage radish contained 78-218 kg N ha-1 (shoot plus root) and early-planted rye contained 43-112 kg N ha-1 by late-fall. In Massachusetts on a Hadley fine sandy loam soil, forage radish planted in late-August to early-September had N accumulation of 128 kg ha-1 (5730 kg ha-1 dry matter) in the root plus shoot, and cereal rye with the same planting dates had N accumulation of 41 kg ha-1 (2650 kg ha-1 dry matter) (Jahanzad, et al., 2017). On a sandy loam in Denmark, forage radish N uptake (158 kg N ha-1) was greater than cereal rye N uptake (91 kg N ha-1) (Kristensen and Thorup-Kristensen, 2004a). 8 Cover crops are often N limited, as evident by their response to N fertilization. Cereal rye N content and biomass was positively correlated to fall N fertilizer application rate (Mirsky, et al., 2017; Komatsuzaki and Wagger, 2015) and residual soil N (Gabriel, et al., 2016; Hashemi, et al., 2013; Ruffo, et al., 2004). In the North Carolina Coastal Plain on a State fine sandy loam soil, the increase in cover crop biomass and N accumulation, with fall ammonium nitrate fertilizer applications compared to no fertilizer, was greater for earlier cover crop planting dates (Komatsuzaki and Wagger, 2015). Manure had a similar effect on cover crops as fertilizer. On a silt loam soil in Pennsylvania and loamy sand soils in Maryland, Ryan, et al. (2011) found that cereal rye biomass increased with poultry litter applications, but biomass did not increase with rye seeding rate. In southern Ontario, forage radish, perennial ryegrass (Lolium perenne L.), and oat (Avena sativus L.) cover crop biomass and N content was significantly higher in fall manured treatments (Thilakarathna, et al., 2015). Cover crops generally did not contain more N in their biomass than the amount applied as fertilizer (Mirsky, et al., 2017; Komatsuzaki and Wagger, 2015; Thilakarathna, et al., 2015). This provides evidence that while cover crops are often N limited, applying fertilizer to cover crops may reduce the overall N use efficiency of the cropping system. Cover crops also respond to N fertilization on the previous corn crop. In Blacksburg, VA on a Hayter silt loam soil, Ditsch, et al. (1993) found that cereal rye biomass and recovery of residual fertilizer N increased with increasing fertilizer N applied to the prior corn crop. In Wisconsin, on a loamy sand soil, Bundy and Andraski (2005) found that cereal rye biomass was significantly higher when N fertilizer was applied to the previous corn crop compared to the corn with no fertilizer. Corn residue N 9 content and the C/N ratio was significantly related to rye biomass (Bundy and Andraski, 2005). Kessavalou and Walters (1997) found that in a corn/soybean rotation, the spring biomass and N uptake of the cereal rye following soybean was influenced by the N rate applied to the corn (approximately two years before). The response of rye biomass and N uptake to the previous year fertilizer rate was positive for one year and negative for one year of the study. (The negative response could not be explained by the authors). The amount of soil N availability at planting can also influence the domination of legume versus non-legume in a bi-culture mix (Möller, et al., 2008; Tribouillois, et al., 2016). The amount and depth of N uptake during the fall months is determined by factors specific to cover crop species including the speed of cover crop establishment and growth, the rooting depth, and the cold tolerance (Thorup-Kristensen, et al., 2003). Nitrogen retention by cover crops was positively correlated with cover crop biomass (R2 = 0.53) and cover crop C/N ratio (R2 = 0.50; Finney, et al., 2016). Planting cover crops earlier in the fall (allowing them to utilizer more growing degree days (GDD)) can significantly increase the capacity of cover crops to accumulate biomass and N across a range of soil types and geographic regions (Hashemi, et al., 2013; Ketterings, et al., 2015; Komainda, et al., 2016; Komainda, et al., 2018; Komatsuzaki and Wagger, 2015; Schroder, et al., 1996; Teixeira, et al., 2016). On a Hagerstown silt loam soil in Pennsylvania, Mirsky, et al. (2011) found that the spring biomass of rye or rye/hairy vetch (Vicia villosa Roth) mix cover crops planted on 25-August was 65% higher than the cover crops planted on 15-October. The loss in cover crop biomass from one date to the next increased through the fall dates (Mirsky, et al., 2011). Farsad, et al. (2011) found that small reductions in GDD had a large negative impact on rye biomass accumulation, 10 and estimated that delaying cover crop planting from the recommended planting date resulted in a 27% decrease in N accumulation for a one week delay, 29% decrease for a two week delay, 66% decrease for a three week delay, and 78% decrease for a four week delay. Vos and Van der Putten (1997) found a strong relationship between cereal rye and forage radish dry matter accumulation and intercepted radiation. Nitrogen uptake by brassica cover crops is more sensitive to growing season than is N uptake by monocots (Thorup-Kristensen, et al., 2003; Lacey and Armstrong, 2015). In a study at Rothamsted and at Woburn Experimental Farms in Bedfordshire, England that investigated winter wheat planting dates, Barraclough and Leigh (1984) found that September-planted wheat had over four times as much root dry weight and root length by March than October- planted wheat. They found for the September planting, roots were present 1 m deep by December, but for the October planting, roots did not reach 1 m until April (Barraclough and Leigh, 1984). Earlier planting also reduces the depth of rooting required to “catch up” with NO3 that is likely to be leaching deeper in the soil profile throughout the fall and winter. Farmers in Maryland typically do not plant cover crops until early- or mid- October (Maryland Department of Agriculture, 2018) after harvesting corn or soybean, often 1-2 months after the corn or soybean roots have stopped taking up NO3 from the soil (Hanway, 1963; Ciampitti, et al., 2013). Root depth and the rate of root growth are important factors determining whether plants are able to acquire N at the times when large amounts of it are available in the soil. Especially in environments with sandier soil or more precipitation, cover crops with fast growing roots may be the optimal system to capture NO3 before it has a chance to leach to soil depths below the root zone. Measures of root depth, root frequency, and root 11 intensity (root intersections m-1 line on minirhizotron) are all highly correlated with subsoil (0.5-1.0 m) NO3 uptake (Thorup-Kristensen, 2001; Thorup-Kristensen, 2006a). Forage radish has been found to grow roots > 2.4 m deep (Kristensen and Thorup- Kristensen, 2004a) and have a depth penetration rate of 2 to 3.5 mm day-1 °C-1 (based on sum of daily average temperatures) (Kristensen and Thorup-Kristensen, 2004a; Thorup- Kristensen, 2001; Smit and Groenwold, 2005). Cereal rye has been found to grow roots 1.15 m deep (Kristensen and Thorup-Kristensen, 2004a) and have a depth penetration rate of 1.2 to 1.7 mm day-1 °C-1 (Kristensen and Thorup-Kristensen, 2004a; Thorup- Kristensen, 2001; Smit and Groenwold, 2005). Forage radish was found to have root frequencies (percentage of 4 x 4 cm crosses where roots observed on minirhizotron) > 40% down to 2.25 m deep (Thorup-Kristensen, 2006a). Forage radish reached 1 m deep with fewer GDD than cereal rye (Kristensen and Thorup-Kristensen, 2004a). While forage radish and winter wheat both reached a depth of approximately 2.5 m, forage radish reached this depth by early-winter but winter wheat did not reach this depth until late-spring (Thorup-Kristensen, et al., 2009). On sandy loam soils in Maryland, in 15-50 cm soil, forage radish had 1.5-2.7 times more roots than cereal rye under highly compacted soil, 1.1-1.9 times more roots than rye under medium compacted soil, and 0.8- 1.2 times more roots than rye under non-compacted soil (Chen and Weil, 2010). Vos, et al. (1998) found that increased soil N supply decreased root length density (cm root cm-3 soil) of rye and forage radish cover crops. Nitrogen uptake by cover crops is correlated with the reduction in NO3-N leaching (Vos and Van Der Putten, 2004; Feyereisen, et al., 2006). A review of literature including studies with a range of soil types, climatic conditions, and tillage practices, 12 found that non-legume cover crops accumulated on average 20-60 kg inorganic N ha-1 and reduced NO3 leaching by on average 70%, in comparison to a no cover crop fallow treatment (Tonitto, et al., 2006). A meta-analysis using eight publications investigating cover crops on the Canterbury Plains of New Zealand (non-legume and legume cover crop species, all experiments on silt loam soils) found that cover crops took up on average 149 kg N ha-1, reduced residual N following the cover crop by 34 kg N ha-1 (57%), and reduced N leaching by 17 kg N ha-1 or 50% (Teixeira, et al., 2016). A meta- analysis from Nordic countries investigating the effects of cover crops interseeding into spring wheat, barley, and oats found that non-legume cover crops reduced fall N leaching loss by 50% and soil inorganic or NO3-N by 35%; legumes did not reduce N leaching (Valkama, et al., 2015). In the Midwest, cover crops have been found to decrease fall and spring residual soil (0-60+ cm deep) NO3-N (Lacey and Armstrong, 2015; Gieske, et al., 2016; Kessavalou and Walters, 1999) and NO3-N leaching (Kaspar, et al., 2007; Strock, et al., 2004). Forage radish proved effective at reducing fall inorganic N, especially in deep soil layers (75-100 cm deep; Thorup-Kristensen, 1994) and reducing leaching (Justes, et al., 1999). In the spring, cereal rye also decreased residual fertilizer-derived N in each 30 cm soil depth increment from 0-90 cm compared to winter fallow (Ditsch, et al., 1993). In Maryland, Dean and Weil (2009) found that forage radish and cereal rye captured nearly all of the NO3 in the soil to 1 m depth, while the no cover crop control plots, particularly in sandy soils, had large pools of NO3 moving down between 60-90 cm. In the fall, the radish cover crop was more effective than cereal rye or rape (Brassica napus L. cv. Dwarf Essex) at depleting NO3 from the soil and taking up N. In the spring, forage radish 13 had higher levels of soil NO3-N from 0-60 cm than cereal rye (Dean and Weil, 2009). In Queen Anne’s County, Maryland, Staver and Brinsfield (1998) found that a cereal rye cover crop following corn reduced annual leaching losses by 80% in comparison to no cover crop. In Beltsville Maryland, a study using tension-drained soil column lysimeters found that NO3 leaching was reduced 95% in dry years and 50% in wet years for cover crops of cereal rye, wheat, or barley (Meisinger and Ricigliano, 2017). Cover crop mixes can have the dual benefit of retaining N with a high-yielding non-legume cover crop, while also supplying N with a legume (Finney, et al., 2016; White, et al., 2017; White, et al., 2016). However, there is concern that including a N- fixing legume within a mixed species cover crop will impede the ability for the cover crop to scavenge soil NO3. For example, prior to 2015, if farmers planted mixed-species cover crops that included a legume, they were not eligible for incentive payments through the Maryland Department of Agriculture cover crop program (Maryland Department of Agriculture, 2015). In order to overcome inherent trade-offs between the retention of N and supply of N by cover crops, cover crop and land management practices can be followed, such as planting cover crop mixtures with low non-legume seeding rates, maintaining low soil NO3-N prior to cover crop planting, utilizing legumes that overwinter, and using non-legumes that are efficient at N retention (White, et al., 2017). Often mixed species cover crops are as effective as monoculture cover crops in reducing residual soil N levels. For example, the amount of NO3 in late-fall in the soil profile (0-90+ cm) was often the same for winter cereal or brassica cover crops with and without legumes, and always less than monoculture legumes (Couëdel, et al., 2018; Möller and Reents, 2009; Tribouillois, et al., 2016). A study using a mix of plant species 14 with different colored roots found that the maximum root depth and depth penetration rate of beet was not affected by the presence of legumes (Tosti and Thorup-Kristensen, 2010). Furthermore, the percent recovery in the fall and spring of surface applied fertilizer for a rye/clover mix was always higher than the clover monoculture and sometimes as high as rye monoculture (Ranells and Wagger, 1997). Cover crops can encourage N mineralization. At a site in North Carolina with a State fine sandy loam soil, Komatsuzaki and Wagger (2015) found that under cereal rye, winter wheat, triticale (Triticum secale L.), and black oats (Avena strigosa L.), the change in soil inorganic N (0-90 cm) between fall and spring sampling dates were correlated with the accumulation of N in the cover crops, and the soil loss was always lower than the cover crop N uptake. They conclude that cover crops are effective N scavengers for both residual soil N, arising for example from previous crop fertilizer, and inorganic N formed from organic N mineralizing during the cover crop season. Alternatively, the change in soil inorganic N between fall and spring sampling dates for the no cover crop treatment (with winter annual weeds) was greater than the weed N accumulation. Cover crops have positive effects on soil microbial abundance and microbial processes (Blanco-Canqui, et al., 2015). Cover crops serve to add substrate for microorganisms throughout their growth through below-ground root exudation and turnover and above-ground leaf litter loss (Thorup-Kristensen, et al., 2003). Furthermore, agricultural practices, such as growing deep-rooted crops, can stimulate the decomposition of organic matter (Fontaine et al, 2007; Kuzyakov, 2010; Schmidt et al, 2011). Cover crop decomposition, nutrient release, and yield of subsequent cash crops 15 The effect of cover crops on the subsequent crop yield varies with factors such as cover crop type, cover crop management (e.g., incorporation date), climatic variables, and the subsequent and previous crop types. Cover cropping cannot be equated to amending the soil, for example through adding manure or compost. Non-legume cover crops do not add N to the soil, but rather capture N from the soil and then return the N back to the soil (Thorup-Kristensen, et al., 2003). Nitrogen that is captured by cover crops can be a valuable resource for farmers, if it is released into the soil as available N in synchrony with cash crop N uptake needs (Dabney, et al., 2001). However, cover crops can have detrimental effects on the environment or agronomic system if cover crop N mineralization leads to increases in N leaching, or if cover crop N immobilization leads to increased fertilizer use on crops (Thorup-Kristensen, et al., 2003). There is sometimes a trade-off between N scavenging and N release. In Slovenia, Kramberger, et al. (2009) found that Italian ryegrass (Lolium multiflorum Lam.) and rape cover crops significantly depleted fall and spring soil inorganic N (0-90 cm), whereas subclover (Trifolium subterraneum L.) and crimson clover decreased soil inorganic N to a lesser extent and less frequently. However, the clovers tended to increase the following corn yield and corn N content, while rape had no effect on corn yield and corn N content, and Italian ryegrass had no effect or decreased corn yield and corn N content. To maximize cover crop N supply and provide the greatest yield benefit to a subsequent corn crop, cover crops should have low C/N ratio and high biomass N content (Finney, et al., 2016; White, et al., 2016). Species that fit these criteria, based on experiments performed in Pennsylvania, include legumes such as fava bean (Vicia faba L.), red clover (Trifolium pretense L.), and hairy vetch, grown in monoculture or in mixtures with each other or with grasses 16 including triticale, Italian ryegrass, or oat or a brassica forage radish. Thomsen, et al. (2016) found during incubation studies with forage radish, white mustard (Sinapis alba L.), and perennial ryegrass using a loamy sand soil that the residue C/N ratio and N concentration were the best single predictors for net N mineralization, regardless of temperature, or of cover crop type, age, or planting date. A meta-analysis including 65 studies (grass included in 47 studies, legume included in 36 studies, mixture included in 13 studies) indicated that corn following a mixed cover crop had 13% higher average yields than corn following no cover crop, corn following a grass cover crop was not different than no cover crop, and corn following a legume cover crop had 21% higher yields than no cover crop (Marcillo and Miguez, 2017). Mixed cover crops with late termination dates (0-6 days before corn planting) had 30% higher corn yield compared to no cover crop (Marcillo and Miguez, 2017). While the corn yield response of cover crops in Canada, and the Great Plains and North Central regions of the USA were not significantly different from yield following no cover crop, the corn yield response in the Southeast and Northeast regions of the USA yielded 12- 14% higher than no cover crop. Cover crops grown in northern regions will have shorter growth seasons and severe winters, which constrain their ability to accumulate biomass and N (Marcillo and Miguez, 2017). Forage radish almost always winter-kills in Maryland and quickly decomposes, releasing inorganic N into the soil surface layers (0-60cm; Dean and Weil, 2009; Lounsbury and Weil, 2014). Jahanzad, et al. (2016) found that by week six of decomposition, as surface residue, forage radish had lost 60% of its initial N concentration while cereal rye had lost 30%. As buried residue, forage radish had lost 17 70% of its initial N concentration, while cereal rye had lost 40%. During the first 12 weeks of decomposition, soil at 20 cm, 40 cm, and 60 cm deep had higher NO3-N concentrations in the radish treatment than rye treatment (Jahanzad, et al., 2017). On a Hadley fine sandy loam soil in Massachusetts, Jahanzad, et al. (2017) found that potato (Solanum tuberosum L.) yield and yield components were higher for potato following forage radish cover crop than cereal rye or no cover crop. Potato grown following forage radish produced the highest yield when fertilized with 75 or 150 kg N ha -1, while potato grown following no cover crop produced the highest yield when fertilized with 225 kg N ha -1 (Jahanzad, et al., 2017). In contrast, studies performed in Minnesota, Wisconsin, and Missouri indicated no fertilizer replacement value or benefit on corn yield of forage radish, despite substantial N uptake by the radish (Gieske, et al., 2016; Ruark, et al., 2018; Sandler, et al., 2015). Nitrogen in winter cereal cover crops is released very slowly by decomposition and is often immobilized by microbes utilizing the abundant carbon in the residues and is therefore largely unavailable for crop uptake (Adeli, et al., 2011; Doran and Smith, 1991; Ketterings, et al., 2015; Thorup‐Kristensen and Dresbøll, 2010). As a result, higher levels of spring N fertilizer are often applied following winter cereal cover crops than would be applied without a cover crop. Several studies have found negative yield responses to winter cereal cover crops. For example, in Iowa, on clay loam and loam soils, corn grain yield was reduced by 6% at the economic optimum N rate, and the negative effect of cereal rye cover crop on corn yield increased with rye cover crop biomass (Pantoja, et al., 2015). Adeli, et al. (2011) found that a cereal rye cover crop decreased cotton (Gossypium hirsutum L.) lint yield in comparison to no cover crop. However, negative 18 responses to rye are not consistent. In a corn/soybean rotation, corn grain yields following rye cover crop were 9.3% lower than yields following no cover crop in only one of the three years of the study, with no differences between yields in the other two years (Kessavalou and Walters, 1997). Kaspar and Bakker (2015) planted wheat, rye, or triticale cover crops before corn in a corn/soybean rotation in Iowa. Cover crops decreased corn yields in two of four years; however effects were different according to cover crop cultivars, and four rye cultivars did not significantly reduce corn yield. Some studies even found a benefit of winter cereal cover crops on following corn yield. In Pennsylvania, on a Hagerstown silt loam soil, Duiker and Curran (2005) found that a cereal rye cover crop did not impact or, if killed-early, may increase corn yields. In Massachusetts on a Hadley fine-sandy loam soil, Hashemi, et al. (2013) found that corn silage (that was not fertilized with N) yield was 34% higher following a cereal rye cover crop and 41% higher following an oat cover crop in comparison to a no cover crop control; however, these cover crops had 64 kg N ha-1 applied to them at planting. Barley following a rye cover crop was found to have higher N supply than following no cover crop if the rye cover crop was incorporated early in the spring and there was heavy winter precipitation. Following dryer winters or later incorporated rye, barley had lower N supply than the no cover crop control (Thorup‐Kristensen and Dresbøll, 2010). The overall consensus from meta-analyses is that negative responses from rye cover crops can be eliminated through management choices. Tonitto, et al. (2006) found that yields of cash crops that were fertilized at the recommended level were no different following non- legume cover crops in comparison to a no cover crop control. A meta-analysis covering 47 studies concluded that grass cover crops had neutral effects on corn yield; however 19 management practices such as the corn N fertilizer rate was a highly significant moderator of yield response (Marcillo and Miguez, 2017). Whereas N credits (extra N available for crop as a result of the cover crop) from grass cover crops are usually negative, requiring that additional fertilizer be applied to the following crop, legume cover crops result in fertilizer credits (reduced fertilizer rates) ranging from 56-135 kg N ha-1 (Doran and Smith, 1991; Meisinger, et al., 1990). Legume cover crops foster microbial N fixation, which adds N to the system. Legume residues also have a relatively low C/N ratio (higher quality residue) which increases N availability following cover crop decomposition. Poffenbarger, et al. (2015a) found that at the end of the corn growing season, cereal rye had released only 8.5 kg N ha -1, while hairy vetch had released 280 kg N ha-1 and a 50/50 mix of rye/vetch had released 139 kg N ha-1. For a hairy vetch/rye mix cover crop, as vetch went from comprising 0 to 100% of the mixture, the N content increased (64 to 181 kg N ha-1) and C/N ratio decreased (83 to 16) (Poffenbarger, et al., 2015b). On sandy loam soils in North Carolina, Wagger (1989b) estimated that in one study after eight weeks of decomposition, rye released 24-26 kg N ha-1, while crimson clover released 73-81 kg N ha-1, and in another study after eight weeks of decomposition, rye released 8-33 kg N ha-1, while crimson clover released 37- 47 kg N ha-1. The N uptake of corn following rye was 21-30 kg N ha-1 less than corn following no cover crop, while the N uptake of corn following crimson clover was 41-45 kg N ha-1 more than corn following no cover crop (Wagger, 1989a). The C/N ratio (Ranells and Wagger, 1997), N content in cover crop residue ( Couëdel, et al., 2018; Seman-Varner, et al., 2017) and corn yield (Clark, et al., 1994) tended to be highest to lower in the order of legume > legume mixed with winter cereal or brassica > winter 20 cereal or brassica monoculture. While some brassica monocultures caused net N immobilization, the brassica/legume mixtures always resulted in net N mineralization. The cover crop mix of brassica/legume served to scavenge N, therein reducing the risk of N leaching, and to provide a green manure, therein reducing the risk of N immobilization and preemptive competition for the subsequent cash crop (Couëdel, et al., 2018). Studies have indicated that N fertilizer can be reduced for a potato crop following legume cover crops in comparison to winter wheat and cereal rye cover crops (Jahanzad, et al., 2017; Sincik, et al., 2008). Preemptive competition can occur, if a cover crop takes up N that would have remained in the rooting zone of the subsequent crop in the absence of the cover crop (Thorup-Kristensen, et al., 2003). While residue mineralization will affect mostly the soil surface layers, which would affect the main crop early in the growing season, preemptive competition of N resources can reduce subsoil N, which could adversely affect the main crop later in the growing season (Thorup-Kristensen, 1993). The apparent effect of cover crops will depend on the soil depth considered. For example, examining 0-50 cm may result in very different conclusions than examining 0-150 cm. To minimize negative preemptive competition effects, the expected leaching intensity of the field and the rooting depth of the subsequent crop should be considered (Thorup-Kristensen and Nielsen, 1998). The long-term goal of using cover crops is to sustain higher levels of production with less N loss, and therefore, the efficacy of cover crops may largely depend on choosing appropriate species according to the local hydrologic regime and minimizing preemptive competition (Thorup-Kristensen, et al., 2003). For example, Thorup- 21 Kristensen (2006a) observed that, in the spring, the subsoil (1-2.5 m) contained 120 kg N ha-1 where no cover crop had been grown but only 49 and 60 kg ha-1, respectively, where radish and Italian ryegrass cover crops had been grown. During the following crop season, they measured the available inorganic N in the root zone for each crop and the actual N uptake by each crop. They found that there was more available N and N uptake for leek (Allium porrum L.) after radish and leek after ryegrass in comparison to leek after no cover crop, and they found there was more available N and N uptake for beet (Beta vulgaris L. var. esculenta L.) after ryegrass (they did not investigate beet after radish) in comparison to beet after no cover crop. However, the N uptake for white cabbage was decreased following ryegrass or forage radish cover crop (Thorup- Kristensen, 2006a). Practical considerations Despite the fact that deeper N (1-2 meters deep) is most at-risk for leaching from the system, most studies only study the topsoil N. Cover crop studies often do not take soil samples deep enough to reveal differences in NO3 depletion and cover crop root growth (Thorup-Kristensen, et al., 2003). Many studies investigating effects of cover crops on soil N (Ebelhar, et al., 1984; Kuo and Jellum, 2002; Ladoni, et al., 2015; Ruffo, et al., 2004; Sainju, et al., 2006) or the effects of other cropping practices on soil N (Anderson and Peterson, 1973; Poudel, et al., 2002; Rice, et al., 1986; Scalise, et al., 2015) have focused on the top 30 cm of soil. There are challenges studying rooting patterns and nutrient uptake by plants deep in the soil. Shallow soil sampling may be due to the difficulty in obtaining deeper soil cores or the misconception that little N exists deeper in the profile and would also be 22 beyond the reach of roots. Deep soil coring is time consuming and laborious. In addition, soils and root systems are more heterogeneous in deeper layers than in topsoil layers. For example, measurements of soil organic carbon (SOC) had a higher coefficient of variation (80.2%) in the subsoil (30-40 cm depth) than in the topsoil (0-10 cm depth) (34.4%) (Usowicz and Lipiec, 2017). In addition, root intensity and root frequency is greatly reduced and therefore more spatially heterogeneous below 1 m deep (Kristensen and Thorup-Kristensen, 2004a). Therefore a greater number of core samples are needed to estimate parameters with confidence, but a smaller number of cores are usually dictated by logistical considerations. Root studies often underestimate root activity by not accounting for fine roots or root turnover with time (Dabney, et al., 2010). Methods of root density (cm cm-3) or intensity (cm cm-2) can differ depending on the methods used (e.g., core break, root wash, minirhizotron methods) (Wahlström, et al., 2015). For example, Wahlström, et al. (2015) found that measurements of forage radish root growth were higher for deeper soil layers using the minirhizotron method than the core break or root wash methods, which was attributed to preferential root growth and root branching along the minirhizotron tube. Nitrogen uptake by individual species within plant mixtures usually cannot be differentiated (Maeght, et al., 2013). Isotopic tracers can be used to assess uptake of applied nutrients from various depths (Hauck and Bremner, 1976; Maeght, et al., 2013). Injecting 15N, a nonradioactive heavy isotope, to a subsurface soil depth is a common method for assessing N uptake by crops or cover crops (Andersen, et al., 2014; Gathumbi, et al., 2003; Ju, et al., 2007; Kristensen and Thorup-Kristensen, 2004a; Kristensen and Thorup-Kristensen, 2004b; Ramirez-Garcia, et al., 2014; Yang, et al., 2014). 23 Cover crops often provide ecosystem services that are cumulative and not immediately measureable, and there is a need for long-term (> 5 year) cover crop studies to better understand the ecosystem services provided by cover crops and the year-to-year variability due to weather (Blanco-Canqui, et al., 2015). Over several years, the use of cover crops can lead to an increase in organic matter to the soil, which can lead to increased mineralization and an increase in plant-available N forms (Hansen, et al., 2000a). In Denmark, on a site with a coarse sand soil and long-term mean precipitation of 868 mm yr-1, Hansen, et al. (2000a) investigated the long-term use of a spring-planted ryegrass cover crop undersown in spring barley or spring wheat. The long-term cover crop treatment had Italian ryegrass or perennial ryegrass grown for the 24 years. They found that long-term use of the cover crop could result in higher NO3 leaching (on average 29% higher) than short-term cover crop use, especially when the cover crop was plowed into the soil in late-fall. Increased NO3 leaching in long-term cover crop systems was accredited to increased mineralization and asynchrony between released cover crop N and crop needs (Hansen, et al., 2000a). Effects of long-term ryegrass use on increased NO3 leaching were evident for at least four years following the discontinuation of cover crop use (Hansen, et al., 2000a). Furthermore, the long-term cover crop resulted in increased wheat yield, and allowed for wheat fertilizer to be reduced with no yield reductions, for over four years following the discontinuation of the cover crop (Hansen, et al., 2000a). A study examining the long-term impact of cover crops over 13+ years in Northern France found that cover crops increased total N stocks from 0-60 cm deep from 11.9-24.2 kg N ha-1 yr-1 (Constantin, et al., 2010). Cover crops also resulted in greater N mineralization and smaller N leaching losses that persisted during the 13-17 year period 24 (Constantin, et al., 2010; Constantin, et al., 2011). Chu, et al. (2017) found that only after three years, soybean yield was 15% higher after a multispecies cover crop mix (grasses, brassicas, clover) compared to a no cover control, while the corn or soybean yields of the first three years following the adoption of the cover crop were not different than the control. Hansen, et al. (2000b) found that introducing a perennial ryegrass cover crop following 25 years of residue removal in a field can cause spring wheat yield increases within two years, resulting in yields similar to the treatment with 24 years of cover crop use. The availability of cheap fertilizer has obviated the need for legume cover crops to provide N nutrition for subsequent cash crops and probably is the main reason for the limited current utilization of cover crops in post-World War II agriculture (Thorup- Kristensen, et al., 2003). A survey of New York dairy farmers found that the primary reasons farmers discontinued the use of cover crops were time requirements and a delay in corn planting, and the primary reasons cited for farmers not adopting cover crop use included lack of time and the perceived high costs of planting cover crops (Long, et al., 2013). Challenges identified through focus groups for adoption of cover crops in Iowa included difficulty in timing of cover crop management (e.g., establishment in the fall and termination in the spring) within corn/soybean rotation systems and costs of establishing and terminating cover crops (Roesch-McNally, et al., 2017). Shorter season corn hybrids could allow farmers to plant cover crops earlier in the fall (Farsad, et al., 2011). Conclusion 25 We hypothesize that considering deep soil N will improve our understanding of plant-soil nutrient cycling dynamics in agricultural systems. Deep soil N is the pool of N most at-risk for leaching and causing environmental problems. However agricultural research typically considers only the top 30-60 cm of soil as relevant to cropping systems. Through increasing our understanding of deep soil N cycling and the relevant environmental or management factors, we could improve the N use efficiency of agricultural systems. Cover crops have been shown to scavenge N to 2+ meters deep. Future work will evaluate various deep-rooted cover crop systems, which could capture and recycle the leftover inorganic N. 26 Chapter 2: Cropland soil profiles in the Mid-Atlantic contain large pools of residual inorganic N Abstract Summer annual crops are either fertilized with large amounts of N (e.g., corn) or they fix large amounts of N (e.g., soybean). In addition, organic matter is releasing N by mineralization during most of the year. We hypothesized that large amounts of mineral N remain in the soil following summer cash crops, particularly in deeper layers. We investigated the amount of mineral N remaining in the soil in September in the Mid- Atlantic USA for 14 fields with Coastal Plain sediment parent materials and 15 fields with Acidic or Calcareous rock parent materials by taking 210 cm deep soil cores. Across the 29 sites, total mineral N in the 0-210 cm profiles ranged from 87.4 to 515 kg N ha-1, with an average of 253 kg ha-1. Of the 253 kg ha-1, 45% was NO3-N and 55% was NH4- N. The soil layers from 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm, contained 22%, 23%, 27%, and 28%, respectively, of the profile mineral N. We took deep soil cores in side-by-side corn-soybean fields in September, and found significantly higher levels of NO3-N following soybean than following corn, but similar levels of NH4-N. The deeper the mineral N is in the profile, the greater the risk that it will leach out of the soil and into groundwater over the winter. The pool of residual deep soil N could serve as a valuable resource for farmers if cover crops could capture and bring it to the surface where it could be recycled to subsequent crops and potentially allow farmers to decrease fertilizer N inputs. 27 Introduction Nitrogen (N) use efficiency in agriculture is a key issue for both maximizing profitability and minimizing environmental impacts of farming. In the Mid-Atlantic region of the USA, N leaching is prevalent, due to the combination of the crops grown, climate, and soils. Corn (Zea mays L.) (for grain or silage) and soybean (Glycine max (L.) Merr.) are the highest land area annual crops in Mid-Atlantic Region (USDA Census of Agriculture, 2012). Corn typically stops taking up N from the soil by early-September (or 100 days after emergence) when corn maturity is approached (Hanway, 1963; Ciampitti, et al., 2013). Furthermore, the region has a humid temperate climate, in which most leaching losses occur during the non-growing season (winter and early-spring), while there is little evapotranspiration (ET) but levels of precipitation equivalent to summer (Meisinger and Delgado, 2002). Excessive N and phosphorus (P) loading in the Chesapeake Bay and its tributaries have caused eutrophication, leading to harmful algal blooms, decreased water clarity, and decreased submerged aquatic vegetation, and periods of hypoxia (dissolved-oxygen concentration < 1.0 mg L-1), stressing and killing aquatic organisms (e.g., shellfish; Phillips and Caughron, 2014; Ator and Denver, 2015). Largely due to environmental concerns related to the Chesapeake Bay, in 1998 the Maryland legislature established the Maryland Water Quality Improvement Act (WQIA), requiring growers to implement nutrient management plans based on N and P (Parker, 2000). Farming operations grossing more than $2500 year-1 must follow approved nutrient management plans, which indicate the nutrient sources (e.g., fertilizer, manure) and amounts that can be added to crops (Maryland Department of Agriculture, 2014). In addition, Chesapeake Bay 28 Watershed Implementation Plans (WIPs) have been developed to indicate how the states in the Chesapeake watershed (Delaware, Maryland, New York, Pennsylvania, Virginia, West Virginia, and the District of Columbia) will meet Total Maximum Daily Load (TMDL) goals of reducing N, P and sediment inputs into the Bay (Environmental Protection Agency, Chesapeake Bay TMDL). A recent evaluation ( Environmental Protection Agency, 2017) concluded that the Agriculture sector in Maryland was not on- track to reach its 2017 water quality target of a 60% reduction from the 2009 N loads into the Bay. Therefore, even with statewide, mandated efforts, N leaching continues to be a concern in Maryland. The spatial and temporal patterns of N in the soil profile influence whether N is accessible to crops during the crop growth period or likely to be lost. Nitrogen remaining at the end of the growing season, especially in deeper soil layers is of particular concern. The deeper the N is in the soil profile, the more likely it is for winter leaching to move it below the root zone of following crops or out of the soil profile and into the groundwater (Thorup-Kristensen, 1994). Many measurements of soil N are only for 30 cm or less depth (Anderson and Peterson, 1973; Chu, et al., 2017; Ebelhar, et al., 1984; Kuo and Jellum, 2002; Ladoni, et al., 2015; Poudel, et al., 2002; Rice, et al., 1986; Sainju, et al., 2006; Scalise, et al., 2015). For example, Poudel, et al. (2002) investigated effects of various farming systems (e.g., with organic vs conventional practices) on soil mineral N, but only looked to 30 cm deep, and Chu, et al. (2017) reported that a multispecies cover crop mix increased soil inorganic N, but only took soil cores to 15 cm deep. Limited information is available on mineral N in deeper soil layers, especially deeper than 1 m. Studies that have investigated deep soil N indicate that even when crops were fertilized at 29 recommended rates, substantial mineral N apparently remained in the soil profile at the end of the crop season. For example, on farms in central and southeastern Pennsylvania, in the fall following corn growth, when N was applied at economic optimum rates, there was on average 74 and 94 kg NO3-N ha -1 in the 0-120 cm soil profile for non-manured and manured sites, respectively (Roth and Fox, 1990). Furthermore, we hypothesized that substantial residual N remains in the soil profile following cash crops due to the performance of early-planted cover crops. Fall cover crops have been documented to capture high amounts of N. For example, in Maryland, Dean and Weil (2009) found that early-planted brassica and rye cover crops captured 36-100 kg N ha-1 and 99-171 kg N ha-1 following corn and soybean, respectively. We therefore saw a need to conduct a survey of farm fields to assess the size of the pool of residual mineral N remaining in Mid-Atlantic cropland soils after summer crop uptake had ceased. Cover crop systems are widely used, especially in Maryland and Delaware due to state-funded cash incentive programs (Maryland Department of Agriculture, 2018). Some cover crops have the potential to scavenge residual N from the soil profile, and could serve as a “catch crop” to capture NO3 in the fall months before it leaches out of reach (Dabney, et al., 2010; Meisinger, et al., 1990; Meisinger, et al., 1991). However, a study from Beltsville, MD found that to scavenge N during fall months, cover crops must be planted by mid-September (chapter three). Currently, cover crops in the region are typically not planted until October or November, and have most of their growth in spring months. We reasoned that these late-planted cover crops would not prevent NO3-N leaching if significant amounts of mineral N remain following cash crops in deeper layers. Investigating the amount and location of residual fall N can allow farmers and 30 policy-makers to make informed decisions about optimal cover cropping practices. For example, if a large pool of mineral N were present in the fall, farmers may be motivated to use early-planted cover crops to capture and gain economic value from some of that N by bringing it to the soil surface. The current study investigated pools of mineral—nitrate-N (NO3-N) and ammonium-N (NH4-N). However, it is important to note that approximately 95-99% of N in soils is in organic forms that are unavailable for plant uptake, and that NO3-N and NH4-N are released from these organic forms during microbial decomposition, which is dependent on various environmental and site specific factors (Dahnke and Johnson, 1990; Weil and Brady, 2017). In addition to being taken up by plants, mineral N forms can be leached, held on clay particles, and lost as gasses (N2, N2O, NO, NH3). In the current study, our specific objectives were: 1) Investigate the amount and depth of mineral-N in soil profiles after summer crop N uptake had ceased in the Mid-Atlantic region. 2) Determine differences between the residual NO3-N and NH4-N in total amounts and depth distribution in the upper 210 cm of soil. 3) Compare pools and depth distribution of residual NO3-N and NH4-N among soils formed from Coastal Plain, Acidic rock, and Calcareous rock parent materials. 4) Compare pools of mineral-N following corn versus following soybean crops. Materials and Methods Location 31 We sampled soil to 210 cm deep on a total of 29 farm fields, on a wide range of commercial farm row-crop fields, in August-September during a three year period (2014- 2016). The timing of the samples was chosen to determine the amount of N left in the profile after summer cash crop N uptake had ceased. Soil was sampled in this survey across the main agricultural regions in Maryland and southeast Pennsylvania, in the Piedmont and Ridge and Valley physiographic regions of Maryland and Pennsylvania and the Coastal Plain physiographic region of Maryland (Figure 1; Table 1). The Coastal Plain region, which extends inland from the Atlantic Ocean and estuaries, tends to be flat and composed primarily of sedimentary rock (Polsky, et al., 2000), having multiple levels of unconsolidated to weakly consolidated acid sands and clays (Ciolkosz, et al., 1989). The Piedmont falls within the foothills of the Appalachian mountain range, to the west of the Coastal Plain. This region is composed primarily of metamorphic and igneous rock (Polsky, et al., 2000). The bedrock is primarily granite and schists, with lowland insets of red shales and sandstones (Ciolkosz, et al., 1989). The Ridge and Valley region falls to the west of the Piedmont, and consists of a folded terrain with several parallel, eroded mountains, and contains mostly sedimentary rock (Polsky, et al., 2000). The bedrock in the ridges is primarily sandstone and in valleys is primarily shale and limestone (Ciolkosz, et al., 1989). The Piedmont terrain is erosional with soils typically less than 1 m deep to rock, having higher clay content and lower sand content than Coastal Plain soils. Piedmont soil infiltration rates are typically 6-15 cm h-1. Coastal Plain soils tend to be deeper with soil infiltration rates of 13-28 cm h-1 (Markewich, et al., 1990). The sandier textures and higher infiltration rates common of many soils in the Coastal Plain region surrounding 32 the Chesapeake Bay allow NO3 to leach more rapidly in comparison to the finer textured soils of the Piedmont areas. The 29 sites were classified into three groups (Table 1), based on soil parent materials: 1) soils formed from coastal plain sediments (Coastal Plain), 2) soils formed from acidic rock parent materials (Acidic), and 3) soils formed from calcareous rock parent materials (Calcareous). In order to provide a representative sample of typical agriculture in the region, most farms practiced no-tillage or limited-tillage, there was a range of manure histories, and sampling on most farms followed corn or soybean crops, although other crops were included, which were common to particular counties (e.g., tobacco). Eight of the 29 fields were selected as four pairs of side-by-side corn and soybean fields, in order to evaluate the effect of previous crop on residual N. The fields that were paired were physically located next to each other and also were in the same soil series. Site descriptions for soil core transect sites, indicating crop, manure, and tillage history, and mapped soil series are given in Table 1. Appendix 1, Table 5 lists soil pH, percent sand, percent clay, percent C, and percent N of the study site soils for each 30 cm depth increment from 0-210 cm, and percent soil organic matter (SOM), and P, K, Mg, Ca, and S (mg kg-1) for 0-30 cm. The study region has a humid climate with annual rainfall relatively uniformly distributed throughout the entire year (Maryland Department of State Planning, 1973). From 1895-1997, the Mid-Atlantic Region had an average annual temperature of 11°C and average monthly precipitation of 87 mm (Polsky, et al., 2000). The climate in the study region varies according to the physiographic configuration, with Coastal Plain 33 average temperatures 1-2° C warmer than Piedmont and Ridge and Valley Maryland temperatures (Planning, 1973). Arrangement of soil cores in surveyed fields In 2014, soil cores were collected along a straight transect going down the slope of the field. Two soil cores were taken at five points along the transect (Figure 2). The two soil cores were spaced 60 cm apart. In 2015, soil cores were taken at four points in the field, one in each of the four blocks of the anticipated future cover crop experiment. At each point, three cores were taken, in three positions relative to the crop stubble—in the row (“row”), 19 cm from the row (“side”), and in the center between two rows (“center”) (Figure 2). Soil was sampled in this way in order to investigate if the position of the soil core, relative to where the N fertilizer may have been applied during June side-dressing, affected the soil N concentrations, in order to address concerns that soil N was being overestimated due to the soil core placement. In order to test for differences between row, side, and center soil core positions, an analysis of variance (ANOVA) was performed with soil core position, soil depth, and the interaction of core position x depth as independent variables and the soil NO3-N or NH4-N as a dependent variable across the seven farms (with rep within farm as the random variable). For soil NO3-N, there was no significant effect for position (p = 0.5594) or position by depth (p = 0.9639). For soil NH4-N, there was no significant effect for position (p = 0.5593) or for position by depth (p = 0.9639). This provided evidence that soil NO3-N or NH4-N was not being overestimated due to sample core placement. 34 In October 2016, soil cores were taken in four sets of side-by-side corn and soybean fields (site identification numbers 14, 15, 16, 17, 26, 27, 28, 29 on Figure 1). The side-by-side corn and soybean fields were sampled on the same day. At some locations, soil samples were taken after corn was harvested but soybean was still in the field (dry and mature). Soil cores were collected along a straight transect going down the slope of the field; two soil cores were taken at five points along the transect. The two soil cores were spaced 75 cm apart. Soil cores were both taken in the crop row between two corn plants or between two soybean plants. Soil sampling and analysis Soil cores were taken by hand driving Veihmeyer probes into the ground using a 6.8 kg drop hammer (Veihmeyer, 1929; Dean and Weil, 2009). Cores were taken from 0 to 210 cm deep when possible, or until the probe hit an impassible layer of rock or hit groundwater. The available equipment and resources did not allow soil cores to be taken deeper than 210 cm. In 2014 and 2016, soil was divided into 15 cm increments and two soil cores taken from each point along the transect were composited for each depth increment. In 2015, soil was divided into 30 cm increments and no cores were composited. Detailed procedures of soil sampling from each year can be found in Appendix 2. The collected soil was put into sealed plastic bags and stored in a cooler with ice for transport to the lab. The soil samples were dried at 40 °C for at least 48 hours, and the soil was sieved through a 2 mm sieve. The weight of the soil at the field moisture level, the weight of the soil after drying, and the weight of the gravel that did not pass through the 2 mm sieve was determined. 35 Exchangeable NO3 and NH4 in the soil was extracted with 0.5 M potassium sulfate (K2SO4) solution. Two grams of dry soil were mixed with 20.0 ml of 0.5 M K2SO4 in 50 ml tubes. The tubes were shaken horizontally at 200 rpm for 30 minutes and then allowed to settle in a vertical position for 10 minutes. The supernatant liquid from the tubes was filtered through VWR 410 filter paper. The filtrate was tested for NO3-N and NH4-N using a Lachat QuikChem 8500 Automated Ion Analyzer (Hach Company, Loveland, CO). The filtrate was analyzed for NH4–N by the salicylate method and for NO2-N and NO3–N by cadmium reduction method. The measured NO3-N and NH4-N (mg NO3-N L -1 or mg NH4-N L -1) was blank-corrected with filtered 0.5 M K2SO4 solution samples and converted to mg NO -N or NH -N kg soil-1 3 4 (Appendix 3). In order to convert values of NO3-N and NH4-N concentrations in the soil to stock amounts of NO3-N and NH4-N in kg ha -1, soil bulk density values were estimated from dry mass of known soil volumes in the cores and corrected for gravel content (Equation 1). The mass and volume of soil was determined for each of the soil cores taken with the Veihmeyer probe. Bulk density values for each farm were based on the average of all cores from that farm for a given depth increment (e.g., 0-120 cm or 120-210 cm) (Appendix 4). Equation 1 Bulk density of soil 𝑔 𝑠𝑜𝑖𝑙 (𝑔 𝑠𝑜𝑖𝑙+ 𝑔𝑟𝑎𝑣𝑒𝑙)−𝑔 𝑔𝑟𝑎𝑣𝑒𝑙 = 𝑐𝑚3 (𝜋𝑟2 𝑔 𝑔𝑟𝑎𝑣𝑒𝑙 ∗ℎ𝑒𝑖𝑔ℎ𝑡)−( ) 2.65 𝑔 𝑐𝑚−3 Where, r = radius (in cm) of soil core, as determined by measuring the inside diameter of soil core tip to three significant figures and dividing by 2. 36 height = length (in cm) of the increment of soil collected estimated bulk density of gravel = 2.65 g cm-3 The pH was analyzed by a glass combination pH electrode and a pH meter (Metler Toledo InLab®413 combination meter). Soil particle size analysis was performed according to the modified pipette method (Gavlak, et al., 2005). Total C and N analysis was performed at University of Maryland Department of Environmental Science and Technology Analytical Lab on LECO CN628 Elemental Analyzer (LECO Corp., St. Joseph, MI; Nelson and Sommers, 1996; Matejovic, 1993). Soil organic matter (SOM) (Loss on Ignition Method) and nutrient content by Mehlich3 extraction (P, K, Mg, Ca, Na, S) was measured at WayPoint Analytical, Inc (Richmond, VA). Statistical analysis All analyses were performed using SAS version 9.4 statistical software (SAS Institute, Cary, NC). The level of probability considered significant was p < 0.05, unless otherwise stated. All ANOVA tests were performed using Proc Mixed. To investigate differences between the residual NH4-N and NO3-N amounts (objective two), an ANOVA was performed for 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm depth increments for all farms, for Coastal Plain farms, for Acidic rock farms, and for Calcareous rock farms, with N-type (NO3-N or NH4-N) as a fixed effect and farm as a random effect. To compare pools of inorganic N among soils formed from Coastal Plain, Acidic rock, and Calcareous rock parent materials (objective three), an ANOVA was performed for each 30 cm increment soil depth for the amount of NO3-N, the amount of NH4-N, and the NO3-N percent of the total mineral N, with parent material group as the fixed effect. To investigate differences in inorganic N among soil depths for each parent 37 material group (objective three), an ANOVA was performed for each parent material group for the amount of NO3-N, the amount of NH4-N, and the NO3-N percent of the total mineral N, with soil depth as a fixed effect and farm as a random effect. To compare pools of inorganic N following corn versus following soybean crops (objective four), for the farms with side-by-side corn and soybean fields, an ANOVA was performed for each 30 cm increment soil depth for the amount of NO3-N and the amount of NH4-N, with crop type (corn or soybean) as the fixed effect and farm as a random effect. A Pearson product-moment correlation was performed using Proc Corr to relate the soil NO3-N, NH4-N, and NO3-N percent of the total mineral N to soil percentages of sand, clay, silt, total C and total N, and the C/N ratio. Results Total mineral N in the 0-210 cm profiles ranged from 87.4 to 515 kg N ha-1 (Figure 3). Across the 29 sites, there was on average of 253 kg ha-1 of mineral N in the upper 210 cm of soil. About 22% of the mineral N was located in the uppermost 30 cm of soil, while another 23% was in the 30 to 90 cm increment. The 90-150 and 150-210 cm increments contained 27% and 28%, respectively, of the profile mineral N (Table 2). Of the average total mineral N, 115 kg N ha-1 was NO3-N and 138 kg N ha -1 was NH4-N. For all layers of Acidic and Calcareous sites and the upper layers of Coastal Plain sites, there were no differences between the amounts of NO3-N and NH4-N. For the Coastal Plain sites, the amount of NO3-N was significantly lower than the amount of NH4-N in the subsoil layers (90-150 cm and 150-210 cm) (Table 2). 38 The distribution of NO3-N among soil depth layers followed different patterns for each of the parent material groups. For the Coastal Plain sites, soil NO3-N was greater from 0-30 cm than all of the other 30 cm depth layers from 30-210 cm. For the Acidic sites, soil NO3-N was greater in the surface soil layer (0-30 cm) and some deep soil layers (120-150 cm, 180-210 cm) than 30-60 cm and/or 60-90 cm layers. For the Calcareous sites, there were no differences in soil NO3-N among soil depth layers. The distribution of NH4-N among soil depth layers followed similar patterns for each parent material groups—the surface layer (0-30 cm) soil had significantly more NH4-N than all deeper layers for Coastal Plain, Acidic, or Calcareous sites, with the exception of 120-150 cm for Acidic sites. The NO3-N percent of the total mineral N was not different among soil depth layers for the Coastal Plain sites or Calcareous sites. For the Acidic sites, the NO3- N percent of the total mineral N was lower for the 0-30 and 30-60 soil depth layers than the 30 cm increment soil depth layers from 90-210 cm (Figure 4). There were differences among parent material groups for the amount of NO3-N and the NO3-N percent of the total mineral N for some soil depth layers, but there were no differences among parent material groups for the amount of NH4-N at any soil depth layer. The Coastal Plain sites had lower soil NO3-N levels than the Acidic sites at 90-120 cm and 120-150 cm depth layers, and than the Calcareous sites at 150-180 cm soil depth. The Coastal Plain sites also had lower NO3-N percent of the total mineral N than the Acidic sites at 90-120 cm, 120-150 cm, and 150-180 cm soil depth layers, and than the Calcareous sites at 120-150 cm, 150-180 cm, and 180-210 cm soil depth layers (Figure 4). 39 We correlated soil percents of sand, clay, silt, total C and total N, and the C/N ratio with the pool sizes of soil NO3-N and NH4-N, and NO3-N percent of the total mineral N in the profiles (Table 3; Table 4). The percent sand was negatively correlated to the NO3-N concentration (p < 0.10) in the 0-30 cm, 90-150 cm, and 150-210 cm soil. In the topsoil layer (0-30 cm), the percent C and percent N were positively correlated (p < 0.05) to soil NO3-N and to the NO3-N percent of the total mineral N. In the 30-90 and 90- 150 cm soil depths, we found a negative correlation (p < 0.1) between pH and NH4-N content, and we found a positive correlation (p < 0.05) between pH and NO3-N percent of the total mineral N. There was significantly more soil NO3-N in September following soybean than following corn in the soil depth increments of 30-60 cm, 120-150 cm, 150-180 cm, and 180-210 cm. The levels of soil NH4-N did not differ between corn or soybean treatments, except in the 180-210 cm soil increment, in which soil NH4-N following soybean was significantly higher than following corn (Figure 5). Discussion We expected surface layers to have higher mineral N, as surface soil layers have the most incorporated plant residues, fertilizer, roots and microbial activity. Soil NH4-N was always higher on surface soil layers than deeper soil layers, and soil NO3-N was higher on surface soil layers than deeper soil layers in some cases. The decomposition and mineralization of surface sources of organic C and N likely resulted in the positive correlations between topsoil percent C or N and the amount of soil NO3-N or the NO3-N percent of the total mineral N. 40 Coastal Plain sites also had less NO3-N in all subsoil layers (30-210 cm deep) than the 0-30 layer, whereas the Acidic and Calcareous sites were more variable. Nitrate- N would be expected to leach more quickly through sandy soils, and we did find that percent sand was negatively correlated with the soil NO3-N concentration (but had no relationship with NH4-N concentration). Across all farms, approximately half of the mineral N was in the NO3-N form and half NH4-N form. It is not uncommon, especially on manured soils, for NH4-N concentrations to be as high or even higher than NO3-N concentrations (Brandi-Dohrn, et al., 1997; Eghball, et al., 2004; Kristensen and Thorup-Kristensen, 2004b; Lacey and Armstrong, 2015; Sainju, et al., 2007). Greater NH4-N levels could be attributed to ammonification exceeding nitrification due to higher soil water content or due to NH4-N retention on clay particle cation exchange sites in the subsoil (Sainju, et al., 2007). Soil NO3-N is assumed to be more transient than soil NH4-N, in that soil NO3-N is accumulating and leaching from the soil each year while soil NH4-N is being retained for multiple years in the soil through cation exchange. However, we did not find a positive correlation between percent clay and NH4-N amounts. This is likely because NH4-N ions are occupying only a small fraction of the cation exchange sites, and therefore all of the soils have clay contents high enough to accumulate NH4-N cations. Ammonium-N levels did not vary among parent material types or between soybean and corn fields (except for at one depth), whereas NO3-N levels varied among parent material types and between corn and soybean crops. Previous studies have found NO3-N levels to be more dynamic than NH4-N levels. Kristensen and Thorup-Kristensen (2004b) found that October residual NO3-N (0-2.5 m profile) varied between crop 41 species, with sweet corn (Zea mays L. Saccharata Koern.) > carrot (Daucus carota L.) > white cabbage (Brassica oleracea L. convar. Capitata), whereas residual NH4-N did not vary between the different species. From soil cores taken in various barley (Hordeum vulgare L.), fescue (Festuca L.), and alfalfa (Medicago sativa L.) cropping systems (samples 1 m deep, six to nine times per year), Bergstrom (1986) found NH4-N did not vary much between treatments, staying between 11 and 13 kg N ha-1, whereas NO3-N ranged between 23 and 68 kg N ha-1. On a silt loam soil and a loamy sand soil in Wisconsin, Bundy, et al. (1993) found that spring soil NO3-N (0-90 cm) was higher following soybean in a corn/soybean rotation than following corn in a no-fertilizer continuous corn rotation, but there was no consistent effect of corn/soybean sequence on NH4-N levels. Soil inorganic N might be expected to increase following corn versus soybean cash crops since corn receives N fertilization, while soybean, a legume, does not usually receive N fertilization. However, we found higher levels of NO3-N following soybean than following corn. Other previous studies have also found corn did not have higher residual soil NO3-N levels than soybean following crop harvest (Jaynes, et al., 2001; Pantoja, et al., 2016; Rembon and MacKenzie, 1997). In Nebraska on a Sharpsburg silty clay loam soil, Kessavalou and Walters (1999) found that May soil residual NO3-N (0- 150 cm) was lower in a continuous corn system than following corn in the corn/soybean rotation system, even though it was fertilized more often (every year) and had 25% less N removed in corn yield than the corn in the corn/soybean rotation. We hypothesize that in well-aerated surface soils, NH4-N released during mineralization is rapidly converted to NO3-N by nitrification, resulting in a high NO3-N 42 percent of the total mineral N where immobilization has not removed the mineral N. This is most evident in comparing the soil mineral N after soybeans versus after corn. Mineral N may be higher following soybean than corn because the soil in a soybean crop is a high N environment with low C/N residues and high N root exudates. Microbial N immobilization, which would remove NO3-N and NH4-N from the soil solution, would be expected to be much lower with soybean residue than with corn residue. Green and Blackmer (1995) found higher N immobilization following corn, due to the larger amount of corn residue than soybean residue, which allowed N to be more available following soybean. Concerning soil acidity, our findings were as expected. We expected that at the lowest pH levels (pH 4-5), nitrification (NH4 transformed to NO3) would be limited, leading to higher NH4 amounts and a lower NO3-N percent of the total mineral N. We also expected that at high pH levels, ammonium could be lost through ammonia volatilization (NH4 transformed to NH3), leading to lower NH4 amounts and a higher NO3-N percent of the total mineral N (Table 4). Conclusions and practical applications Across all sites, 57% (65 kg N ha-1) of NO3-N and 55% (138 kg N ha -1) of total mineral N to 210 cm was located 90-210 cm deep. This large pool of deep soil mineral N remaining after growing corn and soybean poses an environmental risk as the N can leach from the system and pollute bodies of water. It also poses an economic risk if this N is lost to the farmer. On the other hand, if this N was recycled to the surface of the soil where it could provide a substantial amount of N to subsequent crops, it might allow farmers to reduce fertilizer applications. 43 The findings that soil NO3-N was higher following soybean than following corn in much of the soil profile is important for management considerations. Residual soil N is often assumed to be a result of over applying N fertilizer (https://www.npr.org/sections/ thesalt/2017/03/07/518841084/farmers-fight-environmental-regulations), and management practices and policies are primarily concerned with preventing fields that have had fertilizer applications from polluting water sources (Maryland Department of Agriculture, 2014). Legumes such as soybean are not typically fertilized with N, yet our data shows they can leave even more residual N in the soil profile and could pose an even greater risk for water pollution than fertilized crops such as corn. The vertical location of the N is important. Many studies that report effects of cover crops on soil N (Chu, et al., 2017; Ebelhar, et al., 1984; Kuo and Jellum, 2002; Ladoni, et al., 2015; Ruffo, et al., 2004; Sainju, et al., 2006) or other cropping practices on soil N (Anderson and Peterson, 1973; Poudel, et al., 2002; Rice, et al., 1986; Scalise, et al., 2015) after taking 15-30 cm deep soil cores may miss important N patterns in deeper soil layers. Shallow soil sampling may be due to the difficulty in obtaining deeper soil cores and the misconception that N deeper in the profile would be an insignificant amount and/or beyond the reach of roots. However, the deeper N (1-2 meters deep) is most at-risk for leaching from the system. Therefore, practices such as incorporating deep-rooted cover cropping systems into crop rotations should be encouraged in order to scavenge deep soil N before it is lost from the system. 44 Table 1. Site descriptions for soil core transect sites, indicating crop, manure, and tillage history, and soil descriptions. Physiographic regions were determined according to Polsky, et al. (2000). Soil series and phase were determined from Web Soil Survey (WSS) data from USDA NRCS (https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm); soil sample texture was compared to the official soil series descriptions from USDA NRCS (https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/home/?cid=nrcs142p2_053587) to ensure soil samples correlated to the mapping units. Parent material classification based on soil series description and verified with observed soil characteristics (e.g., pH and texture). Most recent Physiographic Site or current region/Parent Sampling Crop rotation Manure Mapped soil Site Name Map vegetation Tillage history material date history history series and phase No. at sampling classification point 2013 no-till, 2012 sludge Ingleside sandy 29 Aug Standing Corn/soybean rotation Occasio incorporated, loam, 2-5% slopes; Caroline I Coastal Plain 24 2014 corn previous 10 years nal 2005-2011 no- Hambrook loam, 0- till, 2004 vertical 2% slopes tillage 28 Aug Standing Unicorn-Sassafras Caroline II Coastal Plain 20 NA1 NA NA 2014 corn loams, 0-2% slopes Perennial Piedmont/ grass (alley Conestoga silt Carroll I 10 1 Sep 2014 NA NA NA Calcareous rocks between loam, 3-8% slopes tomatoes) 29 Aug Standing Nassawango silt Dorchester IA Coastal Plain 22 NA NA NA 2014 corn loam, 0-2% slopes Fallsington sandy Recently 17 Aug loams, 0-2% Dorchester IB Coastal Plain 23 harvested NA NA NA 2015 slopes, Northern wheat Tidewater Area Recently Corn, small gran Regular No-till Ridge and Valley/ 19 Sep Murrill gravelly Franklin I 4 harvested silage, alfalfa Acidic rocks 2014 loam, 8-15% slopes corn 45 Recently Corn, small grain Regular Mostly no-till; Ridge and Valley/ 25 Sep Hagerstown silt Franklin IIB 5 harvested silage Occasional Calcareous rocks 2015 loam, 0-3% slopes corn tillage Recently Piedmont/ Regular Penn loam, 3-8% Frederick I 1 6 Sep 2014 harvested NA NA Acidic rocks slopes corn Recently Piedmont/ Hagerstown loam, Frederick II 2 6 Sep 2014 harvested unknown Regular unknown Calcareous rocks 3-8% slopes corn Regular manure applicati Subsoiled in Recently Double crop Piedmont/ 28 Aug ons 2014, disked Duffield-Ryder silt Frederick IV 3 harvested corn/triticale for > 5 Calcareous rocks 2015 spring once yr-1 until loams, 0-3% slopes corn years and fall 2016 for past 10 years Chester gravelly Recently Piedmont/ 27 Aug silt loam, 3-8% Harford I 11 harvested NA Regular NA Acidic rocks 2014 slopes, moderately corn eroded 2014 corn silage, Recently 2013 corn, 2012 Hatboro-Codorus Piedmont/ Howard IA 12 5 Sep 2014 harvested forage sorghum, 2011 No-till silt loams, 0-3 % Calcareous rocks corn sweet corn, 2010 Occasio slopes sweet corn nal 2015 corn silage, No 2014 soybean, 2013 manure Recently Glenelg loam, 3- Piedmont/ 26 Aug corn grain (rye cover applicati Howard IB 13 harvested No-till 8% slopes; Manor Acidic rocks 2015 crop), 2012 corn ons past corn loam, 8-15% slopes grain, 2011 corn 20+ grain, 2010 corn grain years 2 Piedmont/ 29 Oct Standing 2010-2015 Timothy Gladstone loam, 3 Howard IC 14 manure No-till Acidic rocks 2016 corn hay to 8 percent slopes applicati 46 ons past 10 years 2015 corn silage (rye cover crop), 2014 1 corn, 2013 sorghum, manure Piedmont/ 29 Oct Standing Gladstone loam, 3 Howard ID 15 2012 corn (rye cover applicati No-till Acidic rocks 2016 soybean to 8 percent slopes crop), 2011 soybean, ons past 2010 corn (rye cover 10 years crop) Butlertown- Mattapex silt 28 Aug Standing Kent I Coastal Plain 21 NA NA NA loams, 2-5% 2014 corn slopes, moderately eroded Recently Mattapex fine 11 Sep Kent II Coastal Plain 25 harvested NA NA NA sandy loam, 0-2% 2015 corn slopes Recently Mattapex fine 25 Sep Kent IIB Coastal Plain 26 harvested NA NA NA sandy loam, 0 to 2 2016 corn percent slopes Mattapex fine 25 Sep Standing Kent IIC Coastal Plain 27 NA NA NA sandy loam, 0 to 2 2016 soybean percent slopes Recently Matapeake silt 24 Sep Kent IID Coastal Plain 28 harvested NA NA NA loam, 0 to 2 2016 corn percent slopes Matapeake silt 24 Sep Standing Kent IIE Coastal Plain 29 NA NA NA loam, 0 to 2 2016 soybean percent slopes Recently harvested 2013 pumpkin, 2012 Piedmont/ Occasio No-till past 5+ Glenelg silt loam, Lancaster IA 6 2 Sep 2014 wheat corn, 2011 corn, 2010 Acidic rocks nal years 8-15% slopes followed by soybean cover crop 47 mix (60 cm tall) Recently 2014 pumpkin, 2013 Piedmont/ 12 Sep Occasio No-till past 5+ Glenelg silt loam, Lancaster IB 7 harvested corn, 2012 soybean, Acidic rocks 2015 nal years 3-8% slopes corn 2011 corn Corn silage, forage Mostly no-till Piedmont/ 12 Sep Recent Duffield silt loam, Lancaster V 8 rye, tobacco, alfalfa Regular corn, some no- Calcareous rocks 2015 tobacco 3-8% slopes rotation till tobacco 2013 soybean, 2012 No Recently Collington-Wist Prince 25 Aug corn, 2009-2011 manure No-till past 5+ Coastal Plain 18 harvested complex, 0-2% George’s I 2014 mixed grass hay with ever years corn slopes < 25% legumes applied 2015 wheat double 2011-2013 no- crop soybean, 2014 till; fall 2013 Russett-Christiana Prince 15 Oct Standing corn, 2013 wheat Coastal Plain 16 None chisel plow prior complex, 0 to 2 Georges IIIA 2016 corn double crop soybean, to wheat, 2015- percent slopes 2012 soybean, 2011 2016 no-till corn 2015 wheat double 2011-2013 no- crop soybean, 2014 Recently till; fall 2013 Russett-Christiana Prince 15 Oct soybean, 2013 wheat Coastal Plain 17 harvested None chisel plow prior complex, 0 to 2 Georges IIIB 2016 double crop soybean, soybean to wheat, 2015- percent slopes 2012 soybean, 2011 2016 no-till corn 2011- 2014 no manure; 2011-2014 Sudex in 2004- 2011-2014 no- summer with 20 Aug 2010 till, 2010 and Sassafras loam, 0- St. Mary’s I Coastal Plain 19 Pasture rye/clover in winter, 2014 regular before likely 2% slopes 2010 soybean, 2009 applicati vertical tillage corn ons poultry litter 48 Recently Piedmont/ 20 Sep Chester silt loam, York I 9 harvested Unknown Regular No-till Acidic rocks 2014 3-8% slopes corn 1NA indicates information not available 49 Table 2. Soil NO3-N, NH4-N, and mineral N (NO3-N + NH4-N) (kg N ha -1) for 0-30 cm, 30-90 cm, 90-150 cm, 150-210 cm, and 0-210 cm, and the percent of total mineral N found in each soil depth increment. Values are average of all sites (N=29), Coastal plain sediments sites (N=14), Calcareous rocks sites (N=6), and Acidic rocks sites (N=9). Within a depth increment, values followed by the same lower case letter do not differ significantly. Depth Site increment NO3-N NH4-N Mineral N cm kg N ha -1 (% of 0-210 cm N for depth increment) 0-30 24.9 (22%) a 31.3 (23%) a 56.3 (22%) 30-90 25.2 (22%) a 33.6 (24%) a 58.7 (23%) All site 90-150 30.8 (27%) a 37.0 (27%) a 67.7 (27%) 150-210 33.9 (30%) a 36.0 (26%) a 69.9 (28%) 0-210 115 a 138 a 253 0-30 23.9 (27%) a 30.0 (22%) a 53.9 (24%) Coastal 30-90 23.8 (27%) a 33.5 (24%) a 57.3 (25%) Plain 90-150 20.0 (23%) a 35.7 (26%) b 55.7 (25%) sediments 150-210 20.7 (23%) a 38.1 (28%) b 58.8 (26%) 0-210 88.4 a 137 b 226 0-30 24.1 (18%) a 35.9 (23%) a 60.0 (21%) 30-90 25.2 (19%) a 36.2 (24%) a 61.4 (21%) Acidic rocks 90-150 44.5 (33%) a 43.0 (28%) a 87.5 (30%) 150-210 42.4 (31%) a 38.1 (25%) a 80.5 (28%) 0-210 136 a 153 a 289 0-30 28.5 (20%) a 27.8 (24%) a 56.3 (22%) 30-90 28.1 (19%) a 29.9 (26%) a 58.0 (22%) Calcareous 90-150 35.3 (25%) a 30.9 (27%) a 66.3 (25%) rocks 150-210 52.2 (36%) a 28.0 (24%) a 80.2 (31%) 0-210 144 a 117 a 261 50 Table 3. Twenty-nine farm mean, standard deviation (SD), and range values of soil NO3- N (kg N ha-1), NH -14-N (kg N ha ), and NO3-N percent of the total mineral N (NO3-N + NH4-N), pH, percent sand, clay, and silt, percent total C, percent total N, and C/N ratio. Soil divided into increments of 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm. The percent total N and C/N ratio calculated for 0-30 cm increment only, due to many below detection limit (BDL) N levels in deeper layers. 0-30 cm 30-90cm 90-150cm 150-210 cm Mean SD Range Mean SD Range Mean SD Range Mean SD Range NO3-N 3.15 – 2.22 – 5.40 – 1.77 - -1 24.9 20.6 25.2 17.6 30.8 19.7 33.9 30.2 (kg ha ) 92.0 84.4 70.3 153 NH4-N 11.9 – 12.0 – 9.63 - -1 31.3 14.7 33.6 21.0 37.0 25.3 10.1 - 110 36.0 26.6 (kg ha ) 78.9 96.8 115 NO3-N 0.153- 0.105- 0.163- 0.0810- % of 0.404 0.146 0.419 0.148 0.458 0.145 0.474 0.172 0.757 0.691 0.752 0.818 min N 5.14 – 4.51 – 4.09 – 4.04 – pH 6.14 0.479 5.87 0.725 5.56 0.69 5.44 0.76 7.26 7.40 7.02 7.40 Sand 10.5 – 13.9 – 18.7 – 13.4 – 37.9 13.5 42.0 15.2 55.6 22.5 60.2 24.9 (%) 58.1 70.6 90.1 93.8 Clay 7.71 – 10.8 – 3.63 – 2.31 – 16.7 5.10 23.8 7.99 20.0 13.4 17.1 14.3 (%) 25.2 44.2 53.9 55.4 Silt 27.9 – 14.6 – 3.71 – 3.78 – 45.4 11.2 34.2 11.4 24.4 14.2 22.6 14.3 (%) 72.3 62.2 53.1 47.2 0.351 – 0.133 – 0.0472 – 0.0303 – C (%) 0.897 0.342 0.271 0.207 0.15 0.11 0.12 0.16 1.66 1.23 0.651 0.863 0.0466 – N (%) 0.0919 0.0361 BDL BDL BDL BDL BDL BDL BDL BDL BDL 0.170 5.76 – C:N 9.80 1.63 BDL BDL BDL BDL BDL BDL BDL BDL BDL 14.1 51 Table 4. Correlations coefficient (r) and significance (p-value) for correlations between soil NO3-N (kg N ha -1), NH4-N (kg N ha -1), and NO3-N percent of the total mineral N (NO3-N + NH4-N) with soil percent sand, clay, silt, total C, total N, C/N ratio, and pH. Data for 29 farms analyzed by profile increments of 0-30 cm, 30-90 cm, 90-150 cm, and 150-210 cm. The percent total N and C/N ratio correlated for 0-30 cm increment only, due to N levels below detection limit. Soil texture Percent C and N pH % Sand % Clay % Silt % C % N C/N r -0.38 -0.020 0.46 0.42 0.38 -0.040 0.12 0-30 cm p-value 0.044 0.92 0.012 0.023 0.041 0.84 0.52 r 0.010 -0.067 0.034 0.13 . . 0.086 30-90 cm p-value 0.96 0.729 0.86 0.51 . . 0.66 NO3-N r -0.34 0.23 0.33 0.17 . . -0.078 90-150 cm p-value 0.068 0.23 0.08 0.38 . . 0.69 r -0.40 0.40 0.30 0.026 . . -0.19 150-210 cm p-value 0.03 0.03 0.11 0.89 . . 0.33 r -0.27 0.21 0.23 0.11 0.11 -0.11 -0.25 0-30 cm p-value 0.16 0.28 0.23 0.58 0.58 0.58 0.19 r -0.29 -0.03 0.41 0.062 . . -0.36 30-90 cm p-value 0.13 0.87 0.028 0.75 . . 0.058 NH4-N r -0.097 -0.032 0.18 0.061 . . -0.32 90-150 cm p-value 0.62 0.87 0.34 0.754 . . 0.087 r -0.065 -0.020 0.13 -0.074 . . -0.21 150-210 cm p-value 0.74 0.92 0.49 0.72 . . 0.28 r -0.22 -0.081 0.31 0.38 0.38 -0.13 0.18 0-30 cm p-value 0.24 0.68 0.11 0.040 0.044 0.50 0.35 r 0.18 -0.088 -0.18 0.11 . . 0.43 30-90 cm p-value 0.35 0.65 0.36 0.58 . . 0.019 NO3-N % of min N r -0.20 0.094 0.23 0.12 . . 0.38 90-150 cm p-value 0.30 0.63 0.24 0.52 . . 0.041 r -0.12 0.044 0.16 0.13 . . 0.23 150-210 cm p-value 0.54 0.82 0.40 0.51 . . 0.23 52 Figure 1. Locations in Maryland and Pennsylvania of the 29 crop fields in which a transect of 0-210 cm deep soil cores were taken. 53 Figure 2. Deep soil core placement scheme for (a) 2014 showing placement of all five sets of cores per field, and (b) 2015 showing positions for one of the four sets of cores per field. 54 NO3-N 0-210 500 NH4-N 0-210 400 300 200 100 0 Coastal Plain sediments Acidic rocks Calcareous rocks Figure 3. Twenty-nine farm 0-210 cm NO -N (kg N ha-13 ) and NH4-N (kg N ha -1). Error bars show standard error (SE) of mean. Sites Dorchester IB and Lancaster IB total is for 0-180 cm only. 55 Mineral N in 0-210 cm soil (kg ha-1) Caroline I Caroline II Dorchester IA Dorchester IB Kent I Kent IIB Kent IIC Kent IID Kent IIE Kent IIE Prince Georges I Prince Georges IIIA Prince Georges IIIB St Marys I Franklin I Frederick I Harford I Howard IB Howard IC Howard ID Lancaster IA Lancaster IB York I Carroll I Franklin IIB Frederick II Frederick IV Howard IA Lancaster V Figure 4. Amount of NO3-N and NH4-N (kg N soil layer -1 ha-1) and NO3-N percent of the total mineral N (NO3-N + NH4-N) of each 30 cm depth increment for sites with Coastal Plain sediments, Acidic rocks, and Calcareous rocks parent materials. Different lowercase letters indicate significant differences among depths within each parent material group. Different uppercase letters indicate significant differences among parent material groups within each depth. 56 Figure 5. NO3-N and NH4-N (kg N soil layer -1 ha-1) in four pairs of adjacent corn and soybean fields. The symbols **, *, †, ns indicate p < 0.01, 0.05, 0.1, and not significant. 57 Appendix 1. Transect site soil characteristics Table 5. Study site soil pH, percent sand, percent clay, soil texture, percent C, and percent N for each 15 or 30 cm soil depth increment (0-210 cm), and percent soil organic matter (SOM), P, K, Mg, Ca, and S (mg kg-1) for the upper 30 cm of soil from each site. Each record is the average of two to three composited soil cores from two areas in the field. Data from Dorchester IB 180-210 cm, Lancaster IB 180-210 cm, and St Marys I 195-210 cm is from a single point of a field. Values not determined indicated as nd. Values below detection limit indicated as BDL. 4 Site Depth pH1 Texture Sand2 Clay2 C3 N3 SOM P4 K4 Mg4 Ca4 S4 cm % ppm 0-15 5.7 Sandy loam 59.7 7.3 0.711 0.066 1.55 51.5 59 45 498 6 15-30 5.9 loam 48.7 13.7 0.240 0.027 1.1 5 70 66 474 1 30-45 nd nd nd nd 0.187 BDL -- -- -- -- -- -- 45-60 5.6 Sandy loam 62.1 15.1 0.155 BDL -- -- -- -- -- -- 60-75 nd nd nd nd 0.121 BDL -- -- -- -- -- -- 75-90 5.2 Sandy loam 65.8 17.7 0.071 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.058 BDL -- -- -- -- -- -- Caroline I 105-120 5.2 Sandy loam 73.5 19.0 0.053 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.096 BDL -- -- -- -- -- -- 135-150 5.2 Sandy loam 76.5 18.3 0.067 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.054 BDL -- -- -- -- -- -- 165-180 4.7 Sandy clay 73.9 21.2 0.053 BDL -- -- -- -- -- -- 180-195 nd lnoda m nd nd 0.035 BDL -- -- -- -- -- -- 195-210 4.7 Sandy loam 73.7 18.4 0.087 BDL -- -- -- -- -- -- 0-15 6.3 Sandy loam 56.2 8.8 0.765 0.070 1.9 149 113 64.5 783 9.5 15-30 6.3 Sandy loam 52.7 13.1 0.418 0.037 1.4 61.5 101 73 597 4.5 Caroline II 30-45 nd nd nd nd 0.303 0.031 -- -- -- -- -- -- 45-60 6.2 Sandy loam 59.1 17.5 0.246 BDL -- -- -- -- -- -- 60-75 nd nd nd nd 0.169 BDL -- -- -- -- -- -- 58 75-90 6.3 Sandy loam 82.1 12.2 0.057 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.083 BDL -- -- -- -- -- -- 105-120 6.2 Loamy fine 85.3 8.7 0.053 BDL -- -- -- -- -- -- 120-135 nd nsadn d nd nd 0.132 BDL -- -- -- -- -- -- 135-150 6.2 Loamy fine 81.8 9.4 0.068 BDL -- -- -- -- -- -- 150-165 nd snadn d nd nd 0.064 BDL -- -- -- -- -- -- 165-180 6.2 Loamy fine 87.6 5.8 0.070 BDL -- -- -- -- -- -- 180-195 nd snadn d nd nd 0.045 BDL -- -- -- -- -- -- 195-210 6.3 Fine sand 87.9 5.5 0.054 BDL -- -- -- -- -- -- 0-15 6.0 S ilt loam 22.7 10.6 1.246 0.122 2.85 91 159 136 757 15 15-30 5.8 Silt loam 16.4 17.6 0.614 0.066 1.6 32 96 92.5 558 9 30-45 nd nd nd nd 0.356 0.053 -- -- -- -- -- -- 45-60 5.6 Silt loam 13.0 24.0 0.193 0.037 -- -- -- -- -- -- 60-75 nd nd nd nd 0.142 0.030 -- -- -- -- -- -- 75-90 5.2 loam 44.5 21.1 0.105 0.026 -- -- -- -- -- -- 90-105 nd nd nd nd 0.098 0.024 -- -- -- -- -- -- Dorchester IA 105-120 5.3 Sandy loam 67.4 16.5 0.054 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.308 0.042 -- -- -- -- -- -- 135-150 5.3 Sandy loam 76.5 9.7 0.153 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.084 BDL -- -- -- -- -- -- 165-180 5.3 Loamy fine 81.8 10.4 0.090 BDL -- -- -- -- -- -- 180-195 nd snadn d nd nd 0.050 BDL -- -- -- -- -- -- 195-210 5.3 Loamy fine 81.2 10.0 0.052 BDL -- -- -- -- -- -- 0-30 6.0 sSainltd l oam 34.9 12.8 0.805 0.071 2.25 47.5 43 75.5 643 20 30-60 5.2 loam 35.4 20.0 0.170 BDL - - -- -- -- -- -- Dorchester IB 60-90 4.5 Silt loam 20.1 19.6 0.160 BDL -- -- -- -- -- -- 90-120 4.8 loam 45.7 17.6 0.150 0.025 -- -- -- -- -- -- 120-150 4.9 Sandy loam 59.2 15.3 0.146 BDL -- -- -- -- -- -- 59 150-180 5.1 Sandy loam 62.9 15.2 0.130 BDL -- -- -- -- -- -- 180-210 6.0 loam 27.1 26.9 0.181 0.03 -- -- -- -- -- -- 0-15 5.3 Silt loam 11.2 20.3 0.434 0.061 1.65 7.5 53.5 188 791 44.5 15-30 5.0 Silt loam 9.9 22.3 0.267 0.044 1.45 2.5 45.5 197 692 71 30-45 nd nd nd nd 0.251 0.040 -- -- -- -- -- -- 45-60 4.8 Silt loam 24.4 21.3 0.168 0.029 -- -- -- -- -- -- 60-75 nd nd nd nd 0.115 BDL -- -- -- -- -- -- 75-90 4.8 loam 37.9 20.4 0.316 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.187 BDL -- -- -- -- -- -- Kent I 105-120 4.8 Sandy loam 59.8 15.6 0.097 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.206 0.023 -- -- -- -- -- -- 135-150 5.0 Loamy fine 79.2 6.6 0.141 BDL -- -- -- -- -- -- 150-165 nd nsadn d nd nd 0.084 BDL -- -- -- -- -- -- 165-180 4.9 Loamy fine 85.7 4.1 0.075 BDL -- -- -- -- -- -- 180-195 nd nsadn d nd nd 0.070 BDL -- -- -- -- -- -- 195-210 4.7 Loamy fine 86.0 3.3 0.059 BDL -- -- -- -- -- -- 0-30 6.0 lsoaanmd 51.6 7.7 0.635 0.051 2.65 27.5 72 58 495 2 30-60 6.3 loam 45.7 16.1 0.247 0.030 - - -- -- -- -- -- 60-90 6.3 Loamy fine 81.1 5.6 0.097 BDL -- -- -- -- -- -- Kent II 90-120 6.2 sFainnde sand 92.9 3.6 0.037 BDL -- -- -- -- -- -- 120-150 6.2 Fine sand 87.2 3.7 0.065 BDL -- -- -- -- -- -- 150-180 6.3 Fine sand 94.8 1.5 0.031 BDL -- -- -- -- -- -- 180-210 5.6 Fine sand 92.8 3.1 0.037 BDL -- -- -- -- -- -- 0-15 6.0 loam 45.8 8.2 1.015 0.101 2.1 40 62 98 519 3 15-30 5.7 loam 44.5 10.1 0.509 0.056 1.45 21.5 39.5 48.5 414 2 Kent IIB 30-45 nd nd nd nd 0.292 0.036 -- -- -- -- -- -- 45-60 5.9 loam 36.3 17.9 0.222 0.038 -- -- -- -- -- -- 60-75 nd nd nd nd 0.167 0.033 -- -- -- -- -- -- 60 75-90 5.8 loam 49.3 17.7 0.120 0.027 -- -- -- -- -- -- 90-105 nd nd nd nd 0.067 0.022 -- -- -- -- -- -- 105-120 5.6 Loamy fine 85.0 5.6 0.052 BDL -- -- -- -- -- -- 120-135 nd nsadn d nd nd 0.268 BDL -- -- -- -- -- -- 135-150 5.1 Loamy fine 84.0 3.5 0.052 BDL -- -- -- -- -- -- 150-165 nd nsadn d nd nd 0.050 BDL -- -- -- -- -- -- 165-180 5.3 Fine sand 91.4 3.7 0.038 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.031 BDL -- -- -- -- -- -- 195-210 4.8 Fine sand 92.6 2.7 0.035 BDL -- -- -- -- -- -- 0-15 6.2 loam 50.7 8.4 0.818 0.083 1.85 65.5 81 103 625 7 15-30 6.2 loam 46.4 11.6 0.401 0.042 1.3 25.5 68.5 67.5 541 7.5 30-45 nd nd nd nd 0.281 0.045 -- -- -- -- -- -- 45-60 6.1 loam 47.2 17.9 0.222 0.038 -- -- -- -- -- -- 60-75 nd nd nd nd 0.189 0.031 -- -- -- -- -- -- 75-90 6.0 Sandy loam 58.2 14.2 0.104 0.022 -- -- -- -- -- -- 90-105 nd nd nd nd 0.082 BDL -- -- -- -- -- -- Kent IIC 105-120 6.0 Sandy loam 72.4 9.9 0.078 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.122 BDL -- -- -- -- -- -- 135-150 5.9 Sandy loam 71.4 7.5 0.061 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.056 BDL -- -- -- -- -- -- 165-180 5.7 Loamy fine 83.2 5.2 0.046 BDL -- -- -- -- -- -- 180-195 nd nsadn d nd nd 0.034 BDL -- -- -- -- -- -- 195-210 5.5 Loamy fine 86.9 5.9 0.045 BDL -- -- -- -- -- -- 0-15 6.1 Ssainltd l oam 36.5 12.8 0.984 0.098 2.2 49.5 177 101 551 6 15-30 6.3 Silt loam 28.4 16.3 0.528 0.057 1.75 18.5 76 124 507 8 Kent IID 30-45 nd nd nd nd 0.288 0.039 -- -- -- -- -- -- 45-60 5.6 loam 43.2 21.1 0.200 0.029 -- -- -- -- -- -- 60-75 nd nd nd nd 0.110 BDL -- -- -- -- -- -- 61 75-90 5.5 Sandy loam 81.3 11.4 0.051 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.052 BDL -- -- -- -- -- -- 105-120 5.4 Fine sand 89.4 6.1 0.040 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.060 BDL -- -- -- -- -- -- 135-150 5.1 Fine sand 90.7 6.4 0.037 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.050 BDL -- -- -- -- -- -- 165-180 5.0 Loamy fine 87.9 7.4 0.050 BDL -- -- -- -- -- -- 180-195 nd nsadn d nd nd 0.042 BDL -- -- -- -- -- -- 195-210 5.0 Loamy fine 84.7 7.9 0.047 BDL -- -- -- -- -- -- 0-15 6.3 sSainltd l oam 32.8 14.8 0.848 0.084 2.2 46 113 144 633 9 15-30 6.2 Silt loam 32.8 16.7 0.582 0.063 1.9 23.5 94 142 607 5 30-45 nd nd nd nd 0.284 0.039 -- -- -- -- -- -- 45-60 6.1 loam 46.5 19.1 0.242 BDL -- -- -- -- -- -- 60-75 nd nd nd nd 0.296 BDL -- -- -- -- -- -- 75-90 6.1 Sandy loam 72.7 10.2 0.107 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.084 BDL -- -- -- -- -- -- Kent IIE 105-120 6.1 Loamy fine 86.3 6.8 0.047 BDL -- -- -- -- -- -- 120-135 nd nsadn d nd nd 0.077 BDL -- -- -- -- -- -- 135-150 6.0 Loamy fine 86.4 7.7 0.032 BDL -- -- -- -- -- -- 150-165 nd snadn d nd nd 0.026 BDL -- -- -- -- -- -- 165-180 5.9 Fine sand 88.7 6.3 0.026 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.032 BDL -- -- -- -- -- -- 195-210 6.1 Sandy loam 79.5 11.9 0.037 BDL -- -- -- -- -- -- 0-15 5.2 Sandy loam 59.7 11.9 1.416 0.128 2.9 61 144 165 633 14 15-30 5.2 Sandy loam 56.5 16.0 0.657 0.068 1.85 42 112 152 643 9.5 Prince Georges I 30-45 nd nd nd nd 0.550 0.063 -- -- -- -- -- -- 45-60 4.9 Clay loam 43.4 30.6 0.357 0.052 -- -- -- -- -- -- 60-75 nd nd nd nd 0.193 0.037 -- -- -- -- -- -- 62 75-90 4.6 Sandy clay 55.7 23.9 0.150 0.034 -- -- -- -- -- -- 90-105 nd nloda m nd nd 0.151 0.033 -- -- -- -- -- -- 105-120 4.5 Sandy clay 69.6 20.0 0.187 0.031 -- -- -- -- -- -- 120-135 nd nloda m nd nd 0.353 0.044 -- -- -- -- -- -- 135-150 4.5 Sandy clay 69.6 20.1 0.120 0.028 -- -- -- -- -- -- 150-165 nd lnoda m nd nd 0.103 0.025 -- -- -- -- -- -- 165-180 4.4 Sandy loam 78.3 14.9 0.103 0.023 -- -- -- -- -- -- 180-195 nd nd nd nd 0.091 BDL -- -- -- -- -- -- 195-210 4.4 Sandy loam 77.6 13.6 0.104 0.022 -- -- -- -- -- -- 0-15 6.1 Sandy loam 58.0 9.1 1.038 0.073 2.35 39.5 35.5 89.5 777 4 15-30 5.5 loam 52.0 13.5 0.462 0.033 1.3 7.5 27 52 430 24.5 30-45 nd nd nd nd 0.211 0.029 -- -- -- -- -- -- 45-60 4.7 loam 46.2 22.9 0.146 0.022 -- -- -- -- -- -- 60-75 nd nd nd nd 0.146 0.026 -- -- -- -- -- -- 75-90 4.3 Clay loam 34.1 39.9 0.094 0.029 -- -- -- -- -- -- 90-105 nd nd nd nd 0.092 0.032 -- -- -- -- -- -- Prince Georges IIIA 105-120 4.2 clay 20.6 50.1 0.084 0.033 -- -- -- -- -- -- 120-135 nd nd nd nd 0.100 0.032 -- -- -- -- -- -- 135-150 4.0 clay 16.9 54.5 0.085 0.031 -- -- -- -- -- -- 150-165 nd nd nd nd 0.096 0.032 -- -- -- -- -- -- 165-180 4.0 clay 12.2 55.7 0.064 0.030 -- -- -- -- -- -- 180-195 nd nd nd nd 0.075 0.029 -- -- -- -- -- -- 195-210 4.0 clay 14.6 49.9 0.067 0.031 -- -- -- -- -- -- 0-15 5.8 Sandy loam 54.8 8.8 1.169 0.099 2.5 21.5 41 57 548 2 15-30 5.5 loam 47.3 15.6 0.433 0.041 1.4 5.5 24 44.5 425 11.5 Prince Georges IIIB 30-45 nd nd nd nd 0.215 BDL -- -- -- -- -- -- 45-60 4.6 Clay loam 43.3 32.3 0.120 BDL -- -- -- -- -- -- 60-75 nd nd nd nd 0.176 BDL -- -- -- -- -- -- 63 75-90 4.6 Sandy clay 46.1 29.2 0.093 BDL -- -- -- -- -- -- 90-105 nd nloda m nd nd 0.083 BDL -- -- -- -- -- -- 105-120 4.4 Sandy clay 48.3 29.9 0.072 BDL -- -- -- -- -- -- 120-135 nd lnoda m nd nd 0.120 BDL -- -- -- -- -- -- 135-150 4.5 Sandy clay 51.6 26.7 0.077 BDL -- -- -- -- -- -- 150-165 nd nloda m nd nd 0.179 BDL -- -- -- -- -- -- 165-180 4.5 Sandy clay 47.2 25.8 0.083 BDL -- -- -- -- -- -- 180-195 nd lnoda m nd nd 0.089 BDL -- -- -- -- -- -- 195-210 4.4 clay 22.3 42.0 0.098 0.038 -- -- -- -- -- -- 0-15 6.2 loam 41.9 12.4 0.862 0.082 2.1 149 83 75 664 10 15-30 5.3 loam 33.0 20.7 0.548 0.062 1.8 40 62.5 84 460 5 30-45 nd nd nd nd 0.403 0.056 -- -- -- -- -- -- 45-60 5.7 loam 45.6 23.3 0.300 0.043 -- -- -- -- -- -- 60-75 nd nd nd nd 0.278 BDL -- -- -- -- -- -- 75-90 5.3 Sandy loam 72.4 13.5 0.175 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.101 BDL -- -- -- -- -- -- St Marys I 105-120 5.5 Loamy fine 82.4 12.2 0.087 BDL -- -- -- -- -- -- 120-135 nd snadn d nd nd 0.204 BDL -- -- -- -- -- -- 135-150 5.4 Loamy fine 88.7 7.6 0.098 BDL -- -- -- -- -- -- 150-165 nd nsadn d nd nd 0.063 BDL -- -- -- -- -- -- 165-180 5.5 Fine sand 92.0 4.4 0.054 BDL -- -- -- -- -- -- 180-190 nd nd nd nd 0.042 BDL -- -- -- -- -- -- 195-210 5.1 Fine sand 92.7 3.3 0.041 BDL -- -- -- -- -- -- 0-15 5.5 loam 45.9 18.9 0.943 0.092 2.35 19 69 127 777 18.5 15-30 5.9 loam 46.5 22.4 0.458 0.051 1.65 9.5 56 122 718 15.5 Franklin I 30-45 nd nd nd nd 0.309 0.037 -- -- -- -- -- -- 45-60 5.8 Clay loam 39.0 32.7 0.393 0.046 -- -- -- -- -- -- 60-75 nd nd nd nd 0.183 0.028 -- -- -- -- -- -- 64 75-90 6.5 clay 29.3 40.6 0.153 0.028 -- -- -- -- -- -- 90-105 nd nd nd nd 0.232 0.034 -- -- -- -- -- -- 105-120 5.8 clay 29.2 40.5 0.177 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.211 0.031 -- -- -- -- -- -- 135-150 4.8 Clay loam 26.7 27.9 0.220 0.031 -- -- -- -- -- -- 150-165 nd nd nd nd 0.202 0.032 -- -- -- -- -- -- 165-180 4.9 Clay loam 21.7 29.4 0.118 0.026 -- -- -- -- -- -- 180-195 nd nd nd nd 0.131 0.027 -- -- -- -- -- -- 195-210 4.9 loam 28.3 26.2 0.152 0.025 -- -- -- -- -- -- 0-15 6.4 loam 38.5 16.5 1.097 0.118 2.75 80 71.5 125 948 12 15-30 6.2 loam 41.1 17.4 0.536 0.060 1.85 24.5 48 101 929 12.5 30-45 nd loam nd nd 0.319 0.046 -- -- -- -- -- -- 45-60 6.2 Clay loam 36.3 28.6 0.224 0.036 -- -- -- -- -- -- 60-75 nd nd nd nd 0.150 0.033 -- -- -- -- -- -- 75-90 5.2 Clay loam 32.0 30.9 0.119 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.130 0.027 -- -- -- -- -- -- Frederick I 105-120 4.9 loam 42.7 24.1 0.131 0.030 -- -- -- -- -- -- 120-135 nd nd nd nd 0.183 0.034 -- -- -- -- -- -- 135-150 4.8 loam 50.1 11.3 0.082 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.077 BDL -- -- -- -- -- -- 165-180 4.7 loam 47.2 11.4 0.064 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.081 0.023 -- -- -- -- -- -- 195-210 4.9 loam 49.8 10.4 0.082 BDL -- -- -- -- -- -- 0-15 6.1 Silt loam 26.6 19.5 1.738 0.159 4.3 17 95 133 728 11 15-30 5.7 loam 24.9 25.6 0.760 0.078 2.3 4 59 99 543 29 Harford I 30-45 nd nd nd nd 0.433 0.051 -- -- -- -- -- -- 45-60 5.3 loam 29.5 23.3 0.204 0.032 -- -- -- -- -- -- 60-75 nd nd nd nd 0.157 0.024 -- -- -- -- -- -- 65 75-90 5.1 loam 38.5 16.9 0.107 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.083 BDL -- -- -- -- -- -- 105-120 5.2 loam 36.3 14.7 0.099 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.258 0.032 -- -- -- -- -- -- 135-150 5.2 loam 40.5 9.9 0.118 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.093 BDL -- -- -- -- -- -- 165-180 5.2 loam 44.2 8.1 0.100 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.056 BDL -- -- -- -- -- -- 195-210 5.1 loam 43.2 13.2 0.071 BDL -- -- -- -- -- -- 0-30 6.2 loam 47.8 16.7 0.703 0.071 2.1 16.5 51.5 76 571 16 30-60 6.5 loam 50.7 17.2 0.484 0.048 - - -- -- -- -- -- 60-90 6.4 loam 29.2 23.8 0.364 0.039 -- -- -- -- -- -- Howard IB 90-120 6.1 Clay loam 23.7 28.3 0.207 0.034 -- -- -- -- -- -- 120-150 5.7 loam 30.5 23.9 0.134 0.027 -- -- -- -- -- -- 150-180 5.9 loam 42.0 19.8 0.139 0.023 -- -- -- -- -- -- 180-210 5.9 loam 39.9 19.3 0.157 BDL -- -- -- -- -- -- 0-15 6.0 loam 38.3 21.9 1.206 0.127 3.1 7.5 62 84.5 1042 5 15-30 6.6 loam 36.1 26.1 0.397 0.050 2.2 2 42 91.5 998 1.5 30-45 nd nd nd nd 0.260 0.040 -- -- -- -- -- -- 45-60 6.5 Clay loam 30.8 27.2 0.295 0.044 -- -- -- -- -- -- 60-75 nd nd nd nd 0.261 0.037 -- -- -- -- -- -- 75-90 6.0 loam 41.9 22.5 0.113 BDL -- -- -- -- -- -- Howard IC 90-105 nd nd nd nd 0.087 BDL -- -- -- -- -- -- 105-120 5.8 loam 45.1 18.6 0.092 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.159 BDL -- -- -- -- -- -- 135-150 6.1 loam 44.7 16.1 0.071 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.087 BDL -- -- -- -- -- -- 165-180 5.5 Sandy loam 58.1 13.0 0.066 BDL -- -- -- -- -- -- 66 180-195 nd nd nd nd 0.056 BDL -- -- -- -- -- -- 195-210 5.5 Sandy loam 74.7 5.4 0.061 BDL -- -- -- -- -- -- 0-15 6.4 loam 50.3 15.0 1.195 0.130 3.5 27 54.5 71 1077 5.5 15-30 6.5 Sandy loam 59.0 15.8 0.309 0.043 1.95 7 45 70.5 851 4 30-45 nd nd nd nd 0.153 0.026 -- -- -- -- -- -- 45-60 6.0 Sandy loam 62.6 15.5 0.125 BDL -- -- -- -- -- -- 60-75 nd nd nd nd 0.226 0.033 -- -- -- -- -- -- 75-90 5.6 Sandy loam 63.1 15.0 0.066 BDL -- -- -- -- -- -- 90-105 nd nd nd nd 0.070 BDL -- -- -- -- -- -- Howard ID 105-120 5.7 Sandy loam 58.9 16.5 0.107 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.367 0.043 -- -- -- -- -- -- 135-150 5.6 Sandy loam 68.6 11.4 0.045 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.040 BDL -- -- -- -- -- -- 165-180 5.6 Sandy loam 69.7 10.8 0.038 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.051 BDL -- -- -- -- -- -- 195-210 5.5 Sandy loam 71.3 9.8 0.042 BDL -- -- -- -- -- -- 0-15 6.7 Silt loam 29.2 20.6 1.952 0.202 4.7 87 154 116 1401 29.5 15-30 6.3 loam 33.1 22.6 0.953 0.098 3.1 33.5 47.5 84 841 7.5 30-45 nd nd nd nd 0.286 0.039 -- -- -- -- -- -- 45-60 6.4 Clay loam 35.3 27.3 0.414 0.049 -- -- -- -- -- -- 60-75 nd nd nd nd 0.176 0.029 -- -- -- -- -- -- 75-90 6.4 loam 44.9 23.3 0.090 BDL -- -- -- -- -- -- Lancaster IA 90-105 nd nd nd nd 0.102 BDL -- -- -- -- -- -- 105-120 6.3 Sandy loam 52.8 18.2 0.079 BDL -- -- -- -- -- -- 120-135 nd nd nd nd 0.266 0.038 -- -- -- -- -- -- 135-150 5.9 Sandy loam 55.4 17.6 0.095 BDL -- -- -- -- -- -- 150-165 nd nd nd nd 0.078 BDL -- -- -- -- -- -- 165-180 5.5 Sandy loam 55.5 17.2 0.060 BDL -- -- -- -- -- -- 67 180-195 nd nd nd nd 0.061 BDL -- -- -- -- -- -- 195-210 5.4 Sandy loam 62.5 11.0 0.044 BDL -- -- -- -- -- -- 0-30 6.1 loam 30.5 25.2 0.869 0.087 3.45 81.5 38 60 763 13.5 30-60 6.2 Clay loam 38.6 29.9 0.292 0.034 -- -- -- -- -- -- 60-90 6.1 Sandy clay 51.5 26.5 0.167 BDL -- -- -- -- -- -- Lancaster IB 90-120 5.7 lSoaanmd y loam 55.1 18.4 0.095 BDL -- -- -- -- -- -- 120-150 6.1 Sandy loam 62.3 15.6 0.140 BDL -- -- -- -- -- -- 150-180 6.0 Sandy loam 67.4 8.3 0.084 BDL -- -- -- -- -- -- 180-210 5.7 Sandy loam 73.7 4.4 0.054 BDL -- -- -- -- -- -- 0-15 5.8 Silt loam 26.8 19.1 2.306 0.233 5.95 35 40.5 172 852 17.5 15-30 6.2 loam 29.3 21.0 0.951 0.106 4.05 14.5 23.5 141 701 5.5 30-45 nd nd nd nd 0.485 0.067 -- -- -- -- -- -- 45-60 6.2 Clay loam 31.4 30.5 0.321 0.055 -- -- -- -- -- -- 60-75 nd nd nd nd 0.262 0.049 -- -- -- -- -- -- 75-90 5.9 Clay loam 34.2 34.4 0.191 0.042 -- -- -- -- -- -- 90-105 nd nd nd nd 0.121 0.035 -- -- -- -- -- -- York I 105-120 5.6 Sandy clay 47.6 28.1 0.122 0.036 -- -- -- -- -- -- 120-135 nd nloda m nd nd 0.498 0.066 -- -- -- -- -- -- 135-150 5.3 Sandy clay 50.2 24.9 0.161 0.036 -- -- -- -- -- -- 150-165 nd lnoda m nd nd 0.185 0.036 -- -- -- -- -- -- 165-180 5.5 Sandy loam 57.5 18.6 0.175 BDL -- -- -- -- -- -- 180-195 nd nd nd nd 0.130 0.032 -- -- -- -- -- -- 195-210 5.3 Sandy loam 58.9 19.2 0.165 0.029 -- -- -- -- -- -- 0-15 6.7 loam 36.5 20.8 1.506 0.204 3.65 59 114 51 1368 13 15-30 6.5 loam 40.5 20.3 0.500 0.120 2.15 10.5 64 55 873 12 Carroll I 30-45 nd nd nd nd 0.232 0.090 -- -- -- -- -- -- 45-60 6.4 loam 48.6 17.0 0.198 0.087 -- -- -- -- -- -- 60-75 nd nd nd nd 0.153 0.079 -- -- -- -- -- -- 68 75-90 6.4 loam 49.9 13.6 0.093 0.073 -- -- -- -- -- -- 90-105 nd nd nd nd 0.097 0.075 -- -- -- -- -- -- 105-120 6.0 Sandy loam 52.9 14.1 0.091 0.075 -- -- -- -- -- -- 120-135 nd nd nd nd 0.116 0.077 -- -- -- -- -- -- 135-150 6.0 Sandy loam 54.1 8.5 0.069 0.069 -- -- -- -- -- -- 150-165 nd nd nd nd 0.058 0.072 -- -- -- -- -- -- 165-180 5.5 Sandy loam 58.5 6.2 0.060 0.070 -- -- -- -- -- -- 180-195 nd nd nd nd 0.058 0.075 -- -- -- -- -- -- 195-210 5.4 Sandy loam 65.9 4.1 0.050 0.073 -- -- -- -- -- -- 0-30 6.8 Silt loam 17.9 22.0 1.258 0.120 2.9 49 80.5 111 1421 3.5 30-60 6.7 Silty clay 11.4 33.2 0.588 0.064 -- -- -- -- -- -- 60-90 6.6 Sloialtmy clay 18.4 30.9 0.441 0.049 -- -- -- -- -- -- Franklin IIB 90-120 5.9 clolaaym 30.6 47.8 0.303 0.044 -- -- -- -- -- -- 120-150 5.6 clay 27.9 59.9 0.179 0.048 -- -- -- -- -- -- 150-180 5.1 clay 17.6 54.0 0.141 0.043 -- -- -- -- -- -- 180-210 5.0 clay 13.9 56.8 0.152 0.045 -- -- -- -- -- -- 0-15 6.3 loam 28.2 22.2 0.842 0.101 2.55 22.5 203 71 1021 11 15-30 6.4 Clay loam 30.2 28.2 0.341 0.063 1.8 2.5 47.5 61 1023 5.5 30-45 nd nd nd nd 0.251 0.057 -- -- -- -- -- -- 45-60 6.1 Clay loam 35.8 28.1 0.161 0.048 -- -- -- -- -- -- 60-75 nd nd nd nd 0.213 0.055 -- -- -- -- -- -- 75-90 5.8 Clay loam 36.5 37.5 0.123 0.050 -- -- -- -- -- -- Frederick II 90-105 nd nd nd nd 0.125 0.056 -- -- -- -- -- -- 105-120 5.3 clay 29.6 42.6 0.139 0.056 -- -- -- -- -- -- 120-135 nd nd nd nd 0.239 0.064 -- -- -- -- -- -- 135-150 5.2 Clay loam 29.6 38.0 0.143 0.054 -- -- -- -- -- -- 150-165 nd nd nd nd 0.129 0.053 -- -- -- -- -- -- 165-180 5.7 Clay loam 41.0 32.0 0.321 0.050 -- -- -- -- -- -- 69 180-195 nd nd nd nd 0.118 0.052 -- -- -- -- -- -- 195-210 5.2 Clay loam 39.0 27.6 0.115 0.050 -- -- -- -- -- -- 0-30 7.3 loam 29.1 22.5 1.025 0.105 2.2 69.5 73.5 108 1470 3.5 30-60 7.4 Silty clay 18.7 40.0 0.285 0.041 - - -- -- -- -- -- 60-90 7.4 clay 18.5 48.5 0.223 0.043 -- -- -- -- -- -- Frederick IV 90-120 6.6 clay 21.6 46.2 0.163 0.038 -- -- -- -- -- -- 120-150 6.7 Clay loam 28.3 38.7 0.137 0.033 -- -- -- -- -- -- 150-180 7.1 clay 26.5 45.9 0.169 0.044 -- -- -- -- -- -- 180-210 7.7 Clay loam 28.5 36.7 1.5575 0.039 -- -- -- -- -- -- 0-15 6.5 loam 43.9 12.6 2.193 0.214 4.2 60.5 126 111 1705 17.5 15-30 6.5 loam 47.7 15.7 1.126 0.103 2.6 36.5 59.5 104 1464 16.5 30-45 nd nd nd nd 1.297 0.108 -- -- -- -- -- -- 45-60 6.8 Clay loam 25.5 32.6 1.423 0.111 -- -- -- -- -- -- 60-75 Nd nd nd nd 1.324 0.097 -- -- -- -- -- -- 75-90 6.9 Clay loam 35.3 27.6 0.883 0.062 -- -- -- -- -- -- 90-105 nd nd nd nd 0.682 0.047 -- -- -- -- -- -- Howard IA 105-120 6.6 Silt loam 35.9 10.2 0.724 0.050 -- -- -- -- -- -- 120-135 nd nd nd nd 0.758 0.058 -- -- -- -- -- -- 135-150 6.8 Sandy clay 47.7 24.9 0.438 0.030 -- -- -- -- -- -- 150-165 nd nloda m nd nd 0.374 0.028 -- -- -- -- -- -- 165-180 6.7 loam 48.3 18.6 0.363 0.023 -- -- -- -- -- -- 180-195 nd nd nd nd 0.229 BDL -- -- -- -- -- -- 195-210 7.0 Sandy loam 68.9 12.3 0.256 BDL -- -- -- -- -- -- 0-30 7.1 Silt loam 10.7 17.1 1.581 0.153 2.75 120 134 196 1626 12.5 30-60 6.8 Silt loam 14.0 24.4 0.560 0.055 - - -- -- -- -- -- Lancaster V 60-90 7.0 Silt loam 13.9 23.3 0.352 0.034 -- -- -- -- -- -- 90-120 7.1 Silt loam 17.2 23.6 0.223 0.025 -- -- -- -- -- -- 120-150 7.0 loam 30.9 22.0 0.186 BDL -- -- -- -- -- -- 70 150-180 6.8 loam 26.2 26.4 0.132 BDL -- -- -- -- -- -- 180-210 7.1 Clay loam 24.7 32.0 0.254 0.030 -- -- -- -- -- -- 1 pH by glass combination pH electrode and a pH meter (Metler Toledo InLab®413 combination meter) 2 Soil particle size analysis by the Modified Pipette Method. Gavlak, R., D. Horneck and R.O. Miller. 2005. Particle size analysis modified pipette method. Soil, plant and water reference methods for the western region. 3rd ed. 3 Total C and N analysis at University of Maryland Department of Environmental Science and Technology Analytical Lab (LECO CN628 Elemental Analyzer, LECO Corp., St. Joseph, MI) (Nelson and Sommers, 1996); (Matejovic, 1993). 4 Soil organic matter (SOM; Loss On Ignition method) and nutrient content by Mehlich3 extraction (P, K, Mg, Ca, Na, S) at WayPoint Analytical, Inc (Richmond, VA) 5 Carbon probably originated from inorganic CaCO3 from limestone parent material 71 Appendix 2. Detailed procedures for soil coring Soil cores were taken 0-210 cm deep by hand driving Veihmeyer probes into the ground using a 6.8 kg drop hammer (Veihmeyer, 1929; Dean and Weil, 2009). A 152 cm Veihmeyer probe was used to extract soil from 0-120 cm deep, and a 244 cm or 274 cm Veihmeyer probe was used to extract soil from 120-210 cm deep. After the probe was driven into the ground, a jack and lever system was used to remove the probe from the ground, and the soil was emptied from the probe into a trough (Figure 6). For 2014, 2015, and 2016, the soil coring and division processes differed slightly. In 2014, the 52 cm probe was driven to 120 cm and emptied, extracting soil from 0-120 cm deep, and the 244 cm probe driven to 210 cm and emptied, extracting soil from 120- 210 cm deep. The soil was spread to the appropriate length in the trough—e.g., for the first soil cores it was spread to 120 cm. Soil cores were divided into 15 cm increments. The two soil cores collected from each point along the transect were composited for each depth increment. In 2015, the 152 cm probe was driven to 60 cm and emptied, extracting soil from 0-60 cm deep, the 152 cm probe was driven to 120 cm and emptied, extracting soil from 60-120 cm deep, and the 244 cm probe was driven to 210 cm and emptied, extracting soil from 120-210 cm deep. The soil was spread to the appropriate length in the trough—e.g., for the first soil core it was spread to 60 cm. Soil cores were divided into 30 cm increments. No soil cores were composited. In 2016, the 152 cm probe driven to 60 cm and emptied, extracting soil from 0-60 cm deep, the 152 cm) probe driven to 120 cm and emptied, extracting soil from 60-120 72 cm deep, and the 244 cm) probe driven to 210 cm and emptied, extracting soil from 120- 210 cm deep. The soil was spread to the appropriate length in the trough. Soil cores were divided into 15 cm increments. The two soil cores taken from each point along the transect were composited for each depth increment. 73 Figure 6. Veihmeyer probe, hammer, jack and lever system, and PVC troughs used for taking deep soil cores. 74 Appendix 3. Soil nitrate and ammonium calculations Soil NO3 and NH4 was measured as mg NO3-N L -1 or mg NH -14-N L . Blank samples (samples of filtered 0.5 M K2SO4 solution) were included every 20 soil samples. The average concentration of NO3-N L -1 or NH4-N L -1 in these blank samples was subtracted from the soil sample NO3-N L -1 or NH4-N L -1 for all of the samples analyzed on a particular run of the Lachat instrument, to give the estimated concentration of NO3- N L-1 and NH -14-N L for the sample (Equation 2 shows calculation for NO3-N). The NO3-N L -1 and NH4-N L -1 concentrations were then converted to concentrations in soil (Equation 3 shows calculation for NO3-N), and the amount of NO3-N and NH4-N per area (Equation 4 shows calculation for NO3-N). The NO3-N percent of the total mineral N (NO3-N + NH4-N) was determined. Equation 2 Estimated concentration of NO3-N in extraction solution Estimated mg NO -N L-13 = Measured mg NO -1 3-N L in sample – Average of blank mg NO3-N L -1 Equation 3 Concentrations of NO3-N in soil 𝑚𝑔 𝑁𝑂3−𝑁 𝑚𝑔 𝑁𝑂3−𝑁 20 𝑚𝑙 1 𝐿 1 1000 𝑔 𝑠𝑜𝑖𝑙 = ∗ ∗ ∗ ∗ 𝑘𝑔 𝑠𝑜𝑖𝑙 𝐿 1 1000𝑚𝑙 2 𝑔 𝑠𝑜𝑖𝑙 𝑘𝑔 𝑠𝑜𝑖𝑙 Equation 4 Amount of NO3-N per hectare 𝑘𝑔 𝑁𝑂3−𝑁 𝑚𝑔 𝑁𝑂3−𝑁 1 𝑘𝑔 𝑁𝑂3−𝑁 1 𝑘𝑔 𝑠𝑜𝑖𝑙 𝑔 𝑠𝑜𝑖𝑙 𝑐𝑚 𝑑𝑒𝑝𝑡ℎ 100,000,000 𝑐𝑚 2 = ∗ ∗ ∗ ∗ ∗ ℎ𝑎 𝑘𝑔 𝑠𝑜𝑖𝑙 1000000 𝑚𝑔 𝑁𝑂3−𝑁 1000 𝑔 𝑠𝑜𝑖𝑙 𝑐𝑚 3 1 1 ℎ𝑎 75 Appendix 4. Soil bulk density values The number of cores averaged and the depth increments varied by farm (Table 6). The bulk density values for each layer from each farm were analyzed using box and whisker plots in SAS version 9.4 (SAS Institute, Cary, NC). To exclude outliers and possible errors, values beyond 1.5 x Interquartile range (25th to 75th percentiles) were not included when calculating the average bulk density value. 76 Table 6. Bulk density (BD) mean (g cm-3), standard deviation (SD) (g cm-3), first (Q1) and third (Q3) interquartile range (25th to 75th percentiles) and number of outliers above fences for all farms in which soil cores were taken. The fence is defined as 1.5 x Interquartile range (25th to 75th percentiles). # of BD Soil # of cores BD SD BD BD outliers Site mean (g depth averaged (g cm-3) Q1 Q3 above cm-3) fences Caroline I 0-120 5 1.53 0.273 1.27 1.69 1 120-210 5 1.57 0.0799 1.51 1.65 1 Caroline II 0-120 5 1.36 0.0443 1.33 1.38 1 120-210 5 1.18 0.102 1.17 1.22 1 Carroll I 0-120 5 1.30 0.135 1.21 1.39 1 120-210 5 1.01 0.252 0.836 1.03 1 Dorchester IA 0-120 10 1.55 0.178 1.53 1.68 1 120-210 11 1.22 0.230 1.10 1.40 1 Dorchester IB 0-60 36 1.50 0.125 1.43 1.59 1 60-120 36 1.72 0.213 1.60 1.87 1 120-180 35 1.53 0.297 1.37 1.81 1 Franklin I 0-120 5 1.21 0.147 1.15 1.27 1 120-210 4 1.13 0.385 0.801 1.47 1 Franklin IIB 0-60 11 1.38 0.103 1.29 1.48 1 60-120 12 1.50 0.171 1.37 1.66 1 120-210 6 1.65 0.385 1.53 2.01 1 Frederick I 0-120 29 1.63 0.272 1.54 1.66 1 120-210 25 1.87 0.227 1.71 2.02 1 Frederick II 0-120 5 1.58 0.107 1.50 1.63 1 120-210 5 1.33 0.197 1.14 1.45 1 Frederick IV 0-60 24 1.39 0.0780 1.33 1.47 1 60-120 23 1.538 0.138 1.46 1.63 1 120-210 23 1.38 0.275 1.276 1.62 1 Harford I 0-120 15 1.31 0.273 1.16 1.49 1 120-210 15 1.19 0.362 1.02 1.44 1 Howard IA 0-120 9 1.43 0.0881 1.35 1.49 1 120-210 6 0.985 0.281 0.782 1.28 1 Howard IB 0-60 25 1.308 0.0634 1.26 1.34 1 60-120 25 1.48 0.110 1.38 1.57 1 120-210 27 1.51 0.280 1.38 1.64 1 Kent I 0-120 4 1.16 0.505 0.824 1.50 1 120-210 3 1.17 0.330 0.789 1.40 1 Kent II 0-60 24 1.53 0.124 1.44 1.62 1 60-120 24 1.62 0.0936 1.58 1.66 1 120-210 23 2.08 0.512 1.56 2.65 1 Lancaster IA 0-120 29 1.22 0.217 1.08 1.43 1 120-210 28 1.48 0.240 1.35 1.67 1 Lancaster IB 0-60 26 1.14 0.176 1.01 1.23 1 60-120 25 1.39 0.175 1.28 1.49 1 77 120-210 26 1.44 0.312 1.25 1.57 1 Lancaster V 0-60 40 1.30 0.0814 1.25 1.36 1 60-120 40 1.63 0.115 1.57 1.70 1 120-210 38 1.50 0.323 1.35 1.70 1 Prince Georges I 0-120 5 1.36 0.0716 1.30 1.42 1 120-210 5 1.03 0.184 1.01 1.15 1 St Marys I 0-120 4 1.20 0.0961 1.12 1.28 1 120-210 4 1.48 0.312 1.28 1.69 1 York I 0-120 4 1.12 0.0721 1.08 1.17 1 120-210 4 1.00 0.112 0.922 1.08 1 Howard IC, ID 0-60 10 1.29 0.105 1.22 1.33 1 60-120 10 1.36 0.239 1.17 1.48 1 120-210 10 1.40 0.136 1.27 1.50 1 Kent IIB, IIC 0-60 10 1.56 0.0970 1.51 1.59 1 60-120 10 1.48 0.160 1.36 1.55 1 120-210 10 1.80 0.264 1.63 1.93 1 Kent IID, IIE 0-60 10 1.44 0.126 1.31 1.55 1 60-120 10 1.38 0.212 1.33 1.45 1 120-210 10 1.65 0.102 1.54 1.73 1 Prince Georges IIIA, IIIB 0-60 10 1.29 0.160 1.14 1.40 1 60-120 10 1.37 0.247 1.27 1.45 1 120-210 9 1.74 0.256 1.56 1.80 1 78 Chapter 3: Cover crop species and planting date affect deep soil nitrate capture Abstract Following summer cash crops, substantial mineral N (100-500 kg N ha-1) remains in the 0-2 m soil, which is at risk to leach during the winter and be out of reach for subsequent crops. We hypothesized that cover crops planted by mid-September could capture residual N, and potentially recycle this N to the following cash crop. We buried 15N tracer in deep soil layers (60 cm to 200 cm deep) and evaluated the percent recovery of September- and October-planted cover crops of forage radish, rye, and mixtures of forage radish plus rye, with and without crimson clover. In experiment #1, by December, early-planted cover crops recovered on average 13.7% of the buried 15N from 100 cm deep, while late-planted cover crops recovered only 0.26% from 100 cm deep. In experiment #2, early-planted cover crops recovered on average 14.5% of the buried 15N from 60 cm deep, while the late-planted cover crops recovered only 1.4%. While the percent recovery of the buried 15N from 120 cm and 180 cm deep was low in all cases, the early-planted cover crops recovered more 15N (2.67%) than the late-planted cover crops (0.07%) from 120 cm deep. Early-planted cover crops captured on average 0.31% of the buried 15N from 180 cm deep. We found limited evidence that late-planted cover crops will capture deep soil residual soil N in the spring, but much smaller amounts than the early-planted cover crops capture in the fall. Early-planted radish and rye species, alone or in mixtures, were capable of quick, deep, root growth and are therefore promising “catch-crops”. 79 Introduction Leaching as nitrate (NO3) can be the main pathway for loss of nitrogen (N) from farmland in the Mid-Atlantic USA, especially from November through May when there is little vegetation growing on cropland and precipitation is greater than evapotranspiration (Meisinger, et al., 1991; Shipley, et al., 1992). The uptake of N from the soil by corn (Zea mays L.) typically stops by early September (or 100 days after emergence) when corn maturity is approached (Hanway, 1963; Ciampitti, et al., 2013). At this point the NO3 remaining in the soil and any additional NO3 that is created by mineralization can begin to leach if water is percolating through the soil. In Pennsylvania, on a Hagerstown silt loam soil, 24-55% of fertilizer N applied at economic optimum rates was leached from the soil (Jemison and Fox, 1994). Nitrate leaching poses environmental risks, as NO3 can enter groundwater and bodies of water, such as the Chesapeake Bay. According to the Chesapeake Bay Model, agriculture is responsible for approximately 43% of the N getting into the bay—17% from chemical fertilizer, 19% from manure, and 7% from air deposition of ammonia from livestock (e.g., emissions from poultry houses and dairies) and agricultural soil emissions (Environmental Protenction Agency, 2010). Furthermore, in the Chesapeake Bay watershed, approximately 50% of the N load in streams was transported through groundwater (Phillips and Lindsey, 2013). Excessive N and phosphorus (P) loading in the Chesapeake Bay and its tributaries have caused eutrophication—leading to harmful algal blooms, decreased water clarity, and decreased submerged aquatic vegetation—and periods of hypoxia (dissolved-oxygen concentration < 1.0 mg L-1), stressing and killing aquatic organisms (e.g., shellfish; Ator and Denver, 2015; Phillips and Caughron, 2014). 80 It is important to note that it is desirable to have plentiful N in the soil. Agricultural production is dependent on large pools of available N in the soil profile. However, how deep the N is located is key. Thorup-Kristensen (2006a) noted that the downward leaching of N is not a loss process, but rather the loss occurs if the N leaches beyond the rooting zone of crops. Nitrate that is leaching through the soil profile from August through May will likely be out of reach for the following year’s corn crop. Corn tends to utilize N mostly from soil layers < 50 cm deep, especially in well- fertilized fields (Gass, et al., 1971; Ju, et al., 2007). Corn rooting depth was found to be < 0.8 m at V9 (nine leaf collar) stage and reached a maximum root depth of 1.2 m at silking stage (Zhou, et al., 2008). Some cover crop species have the potential to quickly grow deep roots, and could serve as a “catch crop” to capture NO3 in the fall months before it leaches out of reach, and potentially release N in the spring months to be used by the following cash crop (Dabney, et al., 2010; Meisinger, et al., 1990; Meisinger, et al., 1991). Deep-rooted cover crops could reach deeper soil layers and capture more N from those layers. For example, Zhou, et al. (2008) found that while corn roots did not reach depths > 1.2 m, subsequent winter wheat (Triticum aestivum L.) could use soil NO3 up to 2 m deep. Huang, et al. (1996) found that corn only removed 1 kg NO -1 3-N ha from 120 cm deep soil, whereas switchgrass removed 20 kg NO3-N ha -1. The ability of cover crops to capture deep soil N has been shown to be dependent upon many factors including cover crop species and planting date and soil characteristics. Three main functional groups of cover crops that are grown include winter cereal grasses, legumes, and brassicas (Dabney, et al., 2010). Research has shown that forage radish (Raphanus sativus L. cv. Daikon) and cereal rye (Secale cereale L.) are effective at 81 scavenging deep soil N. Dean and Weil (2009) found that forage radish and cereal rye took up most of the NO3 from the soil profile, while no-cover crop control plots, particularly in sandy soils, had large pools of NO3 moving down between 60-90 cm. In the fall, the radish cover crop was more effective than cereal rye or rape (Brassica napus L. cv. Dwarf Essex) at depleting NO3 from the soil profile and taking up N (Dean and Weil, 2009). Cereal rye is well-documented in its ability to reduce NO3 leaching. Staver and Brinsfield (1998) found that a rye cover crop following corn reduced annual leaching losses by 80% in comparison to no cover crop. In Kentucky, McCracken, et al. (1994) compared NO3 leached over the winter (between corn harvest and corn planting) for cereal rye cover crop versus no cover crop in (NH4)2SO4 fertilized zero-tension lysimeters. Compared to the no cover crop treatment, the rye treatment had 0.2% of the loss of NO3 in year one, 3.6% in year two, and 13.4% in year three (McCracken, et al., 1994). A study using lysimeters in Beltsville, Maryland found that NO3 leaching was reduced 95% in dry years and 50% in wet years for cover crops of cereal rye, wheat, or barley (Hordeum vulgare L.; Meisinger and Ricigliano, 2017). Root depth and the rate of root growth are important factors determining whether plants are able to acquire N at the times when large amounts of it are available in the soil profile. Measures of root depth, root frequency, and root intensity (root intersections m-1 line on minirhizotron) are all highly correlated with subsoil (0.5-1.0 m) NO3 uptake (Thorup-Kristensen, 2001; Thorup-Kristensen, 2006a). Forage radish was found to grow roots > 2.4 m deep, and to have root frequencies (percentage of 4 x 4 cm crosses where roots observed on minirhizotron) > 40% down to 2.25 m deep (Thorup-Kristensen, 2006a; Kristensen and Thorup-Kristensen, 2004a). Cereal rye has been found to grow 82 roots 1.15 m deep (Kristensen and Thorup-Kristensen, 2004a). Forage radish reached 1 m deep with fewer growing degree days than cereal rye (Kristensen and Thorup-Kristensen, 2004a). Planting cover crops earlier in the fall can make a significant difference in the ability of cover crops to capture N. Planting cover crops earlier allows cover crops to utilize more growing degree days. Lacey and Armstrong (2015) found that colder weather conditions likely contributed to less biomass accumulation and N uptake in forage radish compared to cereal rye. Earlier planting also reduces the depth of rooting required to “catch-up” with NO3 that is likely to be leaching deeper in the soil profile throughout the fall and winter. Nitrogen that is captured by cover crops can be a valuable resource for farmers if it is released into the soil as available N in synchrony with cash crop N uptake needs. The release of scavenged N is important for improving the overall N use efficiency of the cropping system. For example, while winter cereals are effective at N scavenging, N in their residues is released very slowly by decomposition and is often immobilized by microbes utilizing the abundant carbon in the residues and is therefore largely unavailable for crop uptake. As a result, higher levels of spring N fertilizer are often applied following winter cereal cover crops than would be applied without a cover crop. Legume cover crops foster microbial N fixation, which adds N to the system. Legume residues also have a relatively low C/N ratio which improves N availability following cover crop decomposition. Legume cover crops result in fertilizer credits (reduced fertilizer rates) ranging from 56-135 kg N ha-1, whereas N credits from grass cover crops are usually negative, requiring that additional fertilizer be applied to the following crop (Doran and 83 Smith, 1991; Meisinger, et al., 1990). Poffenbarger, et al. (2015a) found that at the end of the corn growing season, cereal rye had released only 8.5 kg N ha-1, while hairy vetch (Vicia villosa Roth) had released 280 kg N ha-1 and a 50/50 mix of rye/vetch had released 139 kg ha-1. Kramberger, et al. (2009) found that ryegrass (Lolium multiflorum Lam.) and rape (Brassica napus ssp. Oleifera (Metzg.) Sinsk) cover crops significantly depleted fall and spring soil mineral N in the 0-90 cm soil profile, whereas subclover (Trifolium subterraneum L.) and crimson clover (Trifolium incarnatum L.) decreased soil mineral N to a lesser extent and less frequently; however, the clovers tended to increase the following corn yield and corn N content, while rape had no effect on corn yield and corn N content, and ryegrass had no effect or decreased corn yield and corn N content. Because legumes are N-fixing, there is concern that including a legume within a mixed species cover crop will impede the ability for the cover crop to scavenge soil NO3. For example, prior to 2015, if farmers planted mixed-species cover crops that included a legume, they were not eligible for incentive payments through the Maryland Department of Agriculture cover crop program (Maryland Department of Agriculture, 2015). There are challenges studying rooting patterns and nutrient uptake by plants deep in the soil. Deep soil coring is time-consuming and laborious. In addition, soils and root systems are more heterogeneous in deeper layers than in topsoil layers. For example, measurements of soil organic carbon (SOC) had a higher coefficient of variation (80.2%) in the subsoil (30-40 cm depth) than in the topsoil (0-10 cm depth) (34.4%) (Usowicz and Lipiec, 2017). In addition, root intensity and root frequency is greatly reduced and therefore more spatially heterogeneous below 1 m deep (Kristensen and Thorup- Kristensen, 2004a). Therefore a greater number of core samples are needed to estimate 84 parameters with confidence, but a smaller number of cores are usually dictated by logistical considerations. Root studies often underestimate root activity by not accounting for fine roots or root turnover with time (Dabney, et al., 2010). In addition, N uptake by individual species within plant mixtures usually cannot be differentiated (Maeght, et al., 2013). Isotopic tracers can be used to assess nutrient uptake from various depths (Maeght, et al., 2013). Injecting 15N, a nonradioactive heavy isotope, to a subsurface soil depth is a common method for assessing N uptake by crops or cover crops (Andersen, et al., 2014; Gathumbi, et al., 2003; Ju, et al., 2007; Kristensen and Thorup-Kristensen, 2004a; Kristensen and Thorup-Kristensen, 2004b; Ramirez-Garcia, et al., 2014; Yang, et al., 2014). In the present study, we buried K15NO3 tracer and planted cover crops over the burial points. This allowed us to investigate cover crop uptake from pools of NO3 that were present at a particular depth in late-August. We chose to bury the 15N in late-August because corn in the study region stops taking up soil N at this time. The purpose of this study was to investigate whether NO3 that remains in the subsoil (100 or 200 cm in year one, and 60, 120, or 180 cm in year two) at the end of the corn growing season can be captured by cover crops. Specifically, our objectives were: 1. Evaluate the ability of four cover crops—radish, rye, radish + rye in mix, radish + rye + crimson clover in mix—to capture deep soil NO3; 2. Measure the effect of cover crop planting date on the ability of these cover crops to capture deep soil NO3; 3. Determine if summer corn following no cover crop can capture NO3 that was 60, 120, or 180 cm deep the previous August, and investigate if corn following a 85 cover crop is more likely to contain the NO3 that was 60 cm deep the previous August than corn following no cover crop. Materials and methods Experiment #1 Study sites The study was located at the Central Maryland Research and Education Center— Beltsville Facility in Laurel, Maryland USA. The region has a humid climate with annual rainfall relatively uniformly distributed throughout the entire year. The average annual precipitation for Beltsville, MD is 1063 mm (US Climate Data, 2018). The temperature and precipitation for the study period is found in Figure 7. The experiment was performed from September 2014 to November 2014 during the fall cover crop growing season. The study contained six blocks, three at each of two sites, located 1.30 km away from each other. Site one (39.010638, -76.832985) had a Russett soil, with a loamy fine sand surface horizon texture. Site two (39.018457, -76.820808) had a Christiana soil, with a clay loam surface horizon. Table 7 lists soil pH, percent sand, percent clay, soil texture, percent C, and percent N of the study site soils for each 20 cm depth increment from 0-200 cm, and P, K, Mg, Ca, and S (mg kg-1) for 0-40 cm. Prior to the study, the sites both had wheat with a double crop of soybean in 2014, soybean in 2013, and corn in 2012. Experimental design and treatments The study included 12 treatments in a split-split plot design with three replications at both sites. Plots were 3 m x 3 m in size. Experimental factors that defined the 86 treatments included cover crop planting date, cover crop species, and 15N burial treatments. The treatments were in a complete factorial combination of these factors with cover crop planting date as the main plot factor, cover crop species as the split-plot factor, and 15N burial depth as the split-split plot factor (Figure 8). The cover crop treatment included: 1) forage radish (radish) and 2) cereal rye (rye). Rye is a common cover crop grown in Maryland and the cover crop with the largest monetary incentives under the Maryland cover crop program (Maryland Department of Agriculture, 2018). Radish is a cover crop increasingly being used in Maryland, which has shown much potential for quick root growth and deep N scavenging. The cover crop planting date included: 1) an early-planted date of 28 Aug and 2) a late-planted date of 29 Sep. The 15N burial depth treatments included: 1) 100 cm burial, 2) 200 cm burial, and 3) control treatment in which no 15N was applied. Field operations Double-crop soybeans at reproductive one (R1) stage were mowed (soybean residue remained on field) on 26 Aug 2014 to accommodate the planting date of the study. On 26 Aug 2014, the early-planting date plots were sprayed with Paraquat at the rate of 0.841 kg active ingredient ha-1 for weed control and ammonium sulfate fertilizer at a rate of 22.4 kg N ha-1. Early-planted cover crops were planted 28 Aug 2014. On 27 Sep 2014, the late-planting date plots were sprayed with Paraquat at the rate of 0.841 kg active ingredient ha-1 for weed control and ammonium sulfate fertilizer at a rate of 22.4 kg N ha-1. Late-planted cover crops were planted 29 Sep 2014. All cover crops were planted using a Great Plains Solid Stand 10, no-till drill. 15N solution and burial 87 Solution of 0.5 g KNO3 isotopic tracer 99% enriched in 15N and 250 ml DI (deionized) water were made. Each solution contained 0.07345 g 15N (Equation 5). The 15N solution was buried at one point in the center of each plot. Equation 5 Amount of 15N tracer buried per hole 15.00 g 15N 0.5 g K15NO3 ∗ 15 = 0.07345 g 15N 102.10 g K NO3 After cover crops emerged, the burial point was selected to be in a good stand of cover crop, ideally near center of the 3 m x 3 m plot. A bore hole 7.0 cm in diameter were made vertically to the desired soil depth using a bucket auger, and a 5.1 cm PVC pipe was immediately inserted into the hole. The 250 mL of the K15NO3 solution was poured in the hole, followed by 50 ml of DI (deionized) water to rinse the pipe. Each hole was filled with the removed subsoil to approximately 15 cm above the K15NO3, followed by a 2:1 sand/bentonite mix until the hole was filled up to within 30 cm from the surface. The bentonite mix was used to prevent preferential root growth down the backfilled hole. The top 30 cm of the hole was filled with topsoil from the plot. Cover crop biomass sampling at 15N burial points and preparation for analysis Cover crop biomass was harvested 25 Nov 2014. Weeds were not harvested because they were estimated to be < 5% of biomass. The biomass was harvested if the point where it emerged from the soil fell within a 30 cm radius of the burial point. The radish fleshy taproot (radish root) was pulled from the ground with the leafy top (radish shoot) attached. The root and the shoot were broken apart, and the root was washed. Rye was cut 1 cm above the soil surface. The radish root, radish shoot, and rye tissue types were analyzed separately, as different plant parts may be expected to accumulate 88 different amounts of 15N (Quemada and Cabrera, 1995). Cover crop biomass samples were dried at 40° C, weighed, and ground to < 0.1 mm size. The dry matter per m2 for each tissue type and for each cover crop (sum of tissue types; e.g., radish = radish shoot + radish root) was calculated. Cover crop biomass By 25 Nov 2014, early-planted rye accumulated on average 2500 kg ha-1 dry matter (SE = 160), and early-planted radish shoot accumulated 2600 kg ha-1 dry matter (SE = 180) and root accumulated 1700 kg ha-1 dry matter (SE = 70). Late-planted rye accumulated on average 780 kg ha-1 dry matter (SE = 70), and late-planted radish shoot accumulated 910 kg ha-1 dry matter (SE = 90) and root accumulated 210 kg ha-1 dry matter (SE = 20). Soil sampling at 15N burial points and preparation for analysis In order to investigate NO3 leaching patterns, soil cores were taken 10 cm to the side of the buried 15N tracer on 7 Dec 2014 and 18 May 2015. In December, at site one, a soil core was taken in two of the early-planted rye plots in which the 15N tracer was buried 100 cm deep. Soil cores were taken to 165 cm deep, and the 90-165 cm soil was divided into 15 cm increments. In May, at sites one and two, soil cores were taken in three of the early-planted rye plots in which the 15N tracer was buried 100 cm deep. Soil samples were dried at 40°C for at least 48 hours. The soil was sieved through a 2 mm sieve and then ground to < 0.1 mm. Soil samples from 90-165 cm depths were analyzed for 15N. 15N analysis and calculations 89 Biomass and soil samples were analyzed for 15N at Cornell University Stable Isotope Laboratory using an isotope ratio mass spectrometer (ThermoFinnigan Delta Plus) integrated with an elemental analyzer (Carlo Erba NC2500) through an open split interface (Conflo II). The concentration of 15N in the sample is reported as atom percent (at%) 15N. The amount of 15N uptake per m2 was calculated using Equation 6 and 4. The percent of 15N that was recovered from the total amount buried was calculated using Equation 8 and 6. Equation 6. Tissue type 15N Uptake g 15N uptake of tissue type g dry weight % N at% 15N excesssample = ∗ ∗ m2 m2 100 at% 15N excessfertilizer Where, at% 15N excess = at% 15N − at% 15sample sample Ncontrol at% 15N excess 15 15fertilizer = at% Nfertilizer − at% Ncontrol at% 15Nfertilizer = 99.0 at% 15Ncontrol = average of the 18 control no 15N samples with the same cover crop planting date as the sample Equation 7. Cover crop 15N uptake g 15N uptake 15𝐂𝐨𝐯𝐞𝐫 𝐜𝐫𝐨𝐩 g N uptake𝐓𝐢𝐬𝐬𝐮𝐞 𝐭𝐲𝐩𝐞 = ∑ m2 m2 Where, Radish had two tissue types: 1) radish shoot, 2) radish root Rye had one tissue type: 1) rye Equation 8. Tissue type percent 15N recovery g 1515 N uptake g 15N buried N percent recoveryTissue type = ( / ) ∗ 100 plot area plot area 90 Where, g 15N buried = 0.07345 g Plot area = π * (0.3 m)2 = 0.28 m2 Equation 9. Cover crop 15N percent recovery 15N percent recovery 15Cover crop = ∑ N percent recoveryTissue type Where, Radish had two tissue types: 1) radish shoot, 2) radish root Rye had one tissue type: 1) rye Statistical analysis All analyses were performed using SAS version 9.4 statistical software (SAS Institute, Cary, NC). For all tests, the level of probability considered significant was p < 0.05, unless otherwise stated. We evaluated if cover crops from experimental treatments contained higher at% 15N than background level. The 15N isotope occurs naturally in an almost constant ratio of 1:272 for at% 15N/14N. In other words, 0.366% of N has a mass of 15 rather than 14 (Hauck, et al., 1994). This natural background at% 15N can be compared to the sample at% 15N in order to determine if the sample is enriched or not (Hauck, et al., 1994). The background levels (unenriched) of at% 15N of cover crop tissue were determined by measuring the at% 15N for each cover crop tissue type in control (no 15N) plots. An analysis of variance (ANOVA) was performed to test if the background at% 15N varied due to fixed effects of 1) cover crop tissue type, 2) cover crop planting date, 3) site, 4-7) all interactions of these factors. The site one, rep 3, late-planted, radish 91 root at% 15N value (0.40082) was excluded from the analysis due to being > 5 standard deviations greater than the mean. The ANOVA showed a significant effect (p = 0.0047) for cover crop planting date on the background levels of at% 15N, with early-planting (at% 15N = 0.3699) higher than late-planting (at% 15N = 0.3683). Because there were no differences between sites or among tissue types, the background at% 15N values were pooled over these variables. The at% 15N of the cover crop tissue in an enriched plot was compared to the at% 15N of the cover crop tissue in an unenriched plot (background level) with the same cover crop planting date. A one-sample t-test, which compared a given value to a sample mean, was performed to determine if an experimental treatment value of at% 15N was significantly higher than the background level. Because we were only interested in knowing if cover crops from experimental treatments had a higher at% 15N than the background level (not lower), a “lower one-sided t-test” was performed. The null value (H0) was the at% 15N of the experimental plot in question. The null hypothesis is that the mean of background at% 15N values is equal to the H0; the alternative hypothesis is that the mean of background at% 15N values is less than the H0. Levels of at% 15N significantly above the background at% 15N values were interpreted to mean that the cover crop captured some of the buried 15N tracer. We evaluated how much 15N buried tracer the cover crops captured. An ANOVA was performed to determine if 15N percent recovery was affected by the fixed effects of 1) cover crop, 2) cover crop planting date, 3) 15N burial depth, 4) species x planting date, 5) species x 15N burial depth, 6) planting date x 15N burial depth, 7) species x planting date x 15N burial depth, and 8-14) the interactions of all variables above x site. Random 92 effects were rep(site) and rep x planting date, and rep x species x planting date. The 15N percent recovery response data was not normally distributed so it was log10 transformed, which normalized the distribution. Thus the dependent variable in the ANOVA was log10 transformed 15N percent recovery. Experiment #2 Study sites The study was located at the Central Maryland Research and Education Center— Beltsville Facility in Laurel, Maryland USA. The region has a humid climate with annual rainfall relatively uniformly distributed throughout the entire year. The temperature and precipitation for the study period is found in Figure 9. The experiment was performed from September 2015 to October 2016 during the cover crop-cash crop cycle. The study contained six blocks, three at each of two sites, located 1.06 km away from each other. Site three (39.01162, -76.83167) and site four (39.01837°, -76.82247°) soils were primarily Russett, with a sandy loam surface horizon. Table 8 lists soil pH, percent sand, percent clay, soil texture, percent C, and percent N of the study site soils for each 30 cm depth increment from 0-210 cm, and P, K, Mg, Ca, and S (mg kg-1) for 0-30 cm. Prior to the study, both fields were planted in winter wheat fall 2014-summer 2015. The fields were in corn in 2014, and in fall 2012-summer 2013 the fields were in winter wheat with a double crop of soybean. Experimental design and treatments The study included 29 treatments in a randomized complete block design with six replications. Plots were 3 m x 3 m in size. Experimental factors that defined the treatments included cover crop, cover crop planting date, and 15N burial depth. The 93 treatments were in an incomplete factorial combination of these factors. The cover crop treatment included: 1) radish, 2) rye, 3) radish + rye (two-way mix), 4) radish + rye + crimson clover (three-way mix), and 5) control treatment in which no cover crop was planted. In experiment #2, we added the multi-species cover crop mixture treatments that were not included in experiment #1 in order to investigate how radish and rye perform together versus in monoculture, and to asses if the 15N percent recovery of the rye + radish mix would change with the presence of a legume in the mix. The cover crop planting date treatments included: 1) an early-planting date of 3 Sep and 2) a late-planting date of 8 Oct. There was no late-planting date treatment for the two-way mix, as we did not expect an interaction between planting date and the influence of clover on the cover crop mixture. The 15N burial depth treatments included: 1) 60 cm burial, 2) 120 cm burial, 3) 180 cm burial, and 4) control treatment in which no 15N was applied. There was no 180 cm burial for the late-planting date cover crops, as we did not anticipate cover crops from late-planting reaching even the 120 cm depth (Table 9). Field operations Winter wheat was harvested from the plots mid-July 2015, and weeds were killed on 1 Sep 2015 using Glyphosate, N-(phosphonomethyl)glycine at the rate of 2.31 kg active ingredient ha-1. On 3 Sep 2015, the early-planting date plots were fertilized with 9.07 kg N as urea and ammonium nitrate (UAN), and cover crops were planted. On 28 Sep 2015, the late-planting date plots were fertilized with 9.07 kg N (as UAN). Late- planted cover crops were planted 8 Oct 2015. Cover crops were chemically terminated using Glyphosate, N-(phosphonomethyl) glycine at the rate of 2.31 kg active ingredient ha-1 on 18 May 2016. 94 At site three, corn was planted 16 May 2016. Fertilizer was applied 16 May at planting (45 kg N ha-1) and 14 June 2016 (80.7 N ha-1 and 18.3 kg S ha-1). At site four, corn was planted 7 June 2016. On 8 June 2016, herbicides were applied (1.85 kg ha-1 2- chloro-4-ethylamino-6-isopropylamino-s-triazine (Atrazine), 3.36 kg ha-1 1,1’-dimethyl- 4,4’-bipyridinium dichloride (Paraquat), 1.68 kg ha-1 Acetochlor (Warrant), 0.56 kg ha-1 alcohol ethoxylate, alkylphenol ethoxylate (De-Fac 820 adjuvant non-ionic surfactant). Fertilizer was applied 7 June at planting (45 kg N ha-1) and 29 June 2016 (80.7 N ha-1 and 18.3 kg S ha-1). 15N solution and burial Solutions of 0.5 g KNO3 isotopic tracer 99% enriched in 15N and 250 ml DI (deionized) water were made. Each solution contained 0.07345 g 15N (Equation 5). The 15N tracer was divided among five points per plot. Bore holes 2 cm in diameter were made vertically in the soil using a Veihmeyer probe. Five holes were spatially arranged in the shape of an x, with one hole at the intersection and one at the end of each arm with a distance of 43 cm between the center point and the end of each arm (Figure 10). Immediately following the creation of the five holes in a plot, a PVC pipe was inserted into each hole. Using a funnel, 50 mL of the K15NO3 solution was poured in each of the five holes, followed by 10 ml of DI water to rinse the pipe. A total of 250 mL K15NO3 solution was buried per plot. Each hole was filled with clean quartz sand to approximately 15 cm above the K15NO3, followed by a 2:1 sand/bentonite mix until the hole was filled up to within 30 cm from the surface. The bentonite mix was used to prevent preferential root growth down the backfilled hole. The top 30 cm of the hole was filled with topsoil from the plot. 95 Cover crop biomass sampling at 15N burial points and preparation for analysis We sampled the cover crops with the intention to allow the cover crops to follow their natural process of growth and decomposition as closely as possible. Fall cover crop biomass was sampled between 14 Dec 2015 and 31 Dec 2015. Spring biomass was sampled between 30 April 2016 and 7 May 2016. A plant was sampled if the point where it emerged from the soil fell within a 20 cm radius of each of the five burial points (Figure 10). The tissue types of radish shoot, radish root, rye, and clover shoot were sampled within all treatments. The sampling scheme accounted for the expected differences in C/N ratio and N concentration between the rye and clover stems and leaves (Quemada and Cabrera, 1995). Weeds were not collected because they were estimated to be < 5% of biomass. Radish was expected to naturally winter-kill within one month of the fall sampling date. Therefore we destructively sampled the radish, but returned > 95% of the biomass to the plot to decompose. Radish was harvested by hand-pulling the fleshy root from the soil. The radish root and shoot were separated. The root was thoroughly washed to remove all soil and then weighed. A 4.40 mm diameter bore was taken horizontally through the root (perpendicular) at the vertical midpoint of the root. The root (minus the bore hole) was returned to the exact point in the soil from which it came. The radish shoot was weighed, and split into two equal parts down the midline of the plant. One half of the radish shoot was scattered over the replaced radish root, and the other half of the radish shoot was blended with 30 ml of DI water until liquefied using a food processor. A sample of 30 ml of the blended radish shoot was saved. The remaining radish shoot puree was immediately stored in the refrigerator and scattered within 48 hours over the burial 96 points from the plot harvested. The radish biomass was estimated using percent moisture estimates from a previous study (Equation 10). Equation 10. Estimating radish shoot and root dry matter g dry matter g wet biomass (100 − % moisture) = ∗ m2 m2 100 Where, Wet biomass = values taken in field measurements Percent moisture = values estimated based on a 2012 radish variety trial; average radish shoot percent moisture = 97.07% (N = 20; SD = 1.09), average radish root percent moisture = 93.55% (N = 20; SD = 1.89) (Lounsbury and Weil, unpublished) Rye and clover are expected to overwinter in the study region. Therefore we took minimally-destructive samples of the biomass of these species in the fall and in the spring. For rye, one shoot at the base of the stem was sampled from each clump. A “clump” was defined as all rye leaves coming from a single shoot off a tiller. For clover, one shoot was collected at its base from each clump. A “clump” was defined as all clover stems coming from what appeared to be a single root. To reduce human error, a single investigator identified “clumps” for all plots within a replication. In order to estimate rye biomass, we took measurements of rye patchiness, height, and percent cover. Patchiness was determined by counting the number of clumps. Height was estimated to be the average height of rye leaves within each clump. The leaves were pulled vertical beside a measuring stick to determine the average height. To reduce human error, a single investigator determined the patchiness and height of all plots within a block. To estimate percent cover, a bird’s eye photo was taken of each sampling point; 97 any radish in the plot was removed before the photo was taken. Two investigators made independent visual estimates of percent cover from the photo images; the two estimates were averaged for each point. On areas outside of the plots, we measured rye patchiness, height, and percent cover and then harvested the area and dried and weighed the biomass in order to correlate measurements to actual biomass. We ran a regression analysis to correlate the measured rye parameters to the dry biomass. The analysis selected for the combination of independent variables with the highest adjusted R2. We forced the equation to pass through the origin (i.e., have no y intercept) (Eisenhauer, 2003). Correlation curves were made for each sampling group (Appendix 5, Table 15). The dry matter per m2 for each tissue type and for each cover crop (sum of tissue types; e.g., radish = radish shoot + radish root) was estimated. Crimson clover biomass was not estimated as we did not expect Crimson clover to reach and take up 15N tracer. The average % N for the minimally-destructive and harvested areas outside of the study plots was compared. For the fall samples (N = 20), the ratio of % N from sample to total harvest was on average 0.991, with each sample differing on average 7.86% from the total harvest. For the spring samples (N = 40), the ratio of % N from sample to total harvest was on average 1.01, with each sample differing on average 10.4% from the total harvest. Cover crop biomass December dry matter accumulation for early-planted cover crops was estimated to be 4300 kg ha-1 (SE = 270) for two-way mix (radish shoot and root, rye), 4000 kg ha-1 (SE = 230) for three-way mix (radish shoot and root, rye, excluding clover), 3900 kg ha-1 (SE = 330) for radish (shoot and root), and 1900 kg ha-1 (SE = 90) for rye. December dry 98 matter accumulation for late-planted cover crops was estimated to be 400 kg ha-1 (SE = 40) for three-way mix (radish shoot and root, rye, excluding clover), 460 kg ha-1 (SE = 50) for radish (shoot and root), and 990 kg ha-1 (SE = 70) for rye. Corn sampling at 15N burial points and preparation for analysis Corn plants were sampled when plants had reached the five leaf collar growth stage (V5), on 8 Jun 2016 (for site three) and on 29 Jun 2016 (for site four). Corn grain was sampled on 2 Sep 2016 (for site three) and on 7 Oct 2016 (for site four). Corn was sampled from a 1.8012 m2 circular area (0.7572 cm radius) encompassing the five sampling points. The sampling area was split in half; the corn from one half was harvested at V5 stage and the corn from the other half was harvested for grain. The corn biomass was dried at 40° C and weighed. The dried biomass was ground to < 0.1 mm size. Soil sampling at 15N burial points and preparation for analysis In order to investigate NO3 leaching patterns, soil cores were taken at either 10 cm or 20 cm distance to the side of the buried 15N at several times during the year in the plots that had no cover crops (Table 10). The first set of samples was taken at site three on 20 February 2016 and at site four on 10 April 2016. We intended to take these soil cores in late-fall, but were delayed until February and April by weather and field soil conditions. Soil cores were taken to 210 cm deep and divided into 15 cm increments. Soil samples were dried at 40°C for at least 48 hours. The soil was sieved through a 2 mm sieve and then ground to < 0.1 mm size. We analyzed soil samples from selected depths for at% 15N (Table 10). 15N analysis and calculations 99 Biomass and soil samples were analyzed for 15N at Cornell University Stable Isotope Laboratory using an isotope ratio mass spectrometer (ThermoFinnigan Delta Plus) integrated with an elemental analyzer (Carlo Erba NC2500) through an open split interface (Conflo II). The 15N is reported as atom at% 15N. The 15N uptake was calculated using Equation 11 and 9, and the 15N percent recovery was calculated using Equation 13 and 11. Equation 11. Tissue type 15N uptake g 15N uptake of Tissue type g dry weight % N at% 15N excesssample = ∗ ∗ m2 m2 100 at% 15 N excessfertilizer Where, at% 15N excess 15 15sample = at% Nsample − at% Ncontrol at% 15N excess 15 15fertilizer = at% Nfertilizer − at% Ncontrol at% 15Nfertilizer = 99.0 at% 15Ncontrol = average of the three control no 15N samples with the same tissue type and site as the sample Equation 12. Cover crop 15N uptake g 15N uptake 15𝐂𝐨𝐯𝐞𝐫 𝐜𝐫𝐨𝐩 g N uptakeTissue type = ∑ m2 m2 Where, Radish had two tissue types: 1) radish shoot, 2) radish root Rye had one tissue type: 1) rye shoot Two-way mix had three tissue types: 1) radish shoot, 2) radish root, 3) rye shoot 100 Three-way mix had four tissue types: 1) radish shoot, 2) radish root, 3) rye shoot, 4) clover shoot Equation 13. Tissue type 15N percent recovery 15 15 15 g N uptake g N buried N percent recoveryTissue type = / plot area plot area Where, for cover crop species, g 15N buried = 0.07345 g Plot area = 5*(π (0.2 m)2 = 0.62832 m2 for corn V5 plants or grain, 15.00 g 15N g 15N buried = 0.25 g K15NO3 ∗ = 0.03673 g 102.10 g K15NO3 Plot area = 0.5*(π (0.7572 m)2 = 0.9006 m2 Equation 14. Cover crop 15N percent recovery 15N percent recovery = ∑ 15Cover crop N percent recoveryTissue type Where, Radish had two tissue types: 1) radish shoot, 2) radish root Rye had one tissue type: 1) rye shoot Two-way mix had three tissue types: 1) radish shoot, 2) radish root, 3) rye shoot Three-way mix had four tissue types: 1) radish shoot, 2) radish root, 3) rye shoot, 4) clover shoot Statistical analysis 101 All analyses were performed using SAS version 9.4 statistical software (SAS Institute, Cary, NC). For all tests, the level of probability considered significant was p < 0.05, unless otherwise stated. We evaluated if cover crops from experimental treatments contain higher at% 15N than background level. The background levels (unenriched) of at% 15N of cover crop tissue were determined by measuring the at% 15N for each cover crop tissue type in control (no 15N) plots. An ANOVA was performed to test if the background at% 15N varied due to fixed effects of 1) cover crop tissue type, 2) site, and 3) tissue type x site. Due to the incomplete factorial design, data from early-planted plots was analyzed separately from data from late-planted plots. Fall and spring biomass samples were analyzed separately. For the fall, early-planted cover crops, there were significant effects for site (p = 0.0179) and tissue type (p = 0.0008) on the background levels of at% 15N. Site four (at% 15N = 0.3693) was significantly higher than site three (at% 15N = 0.3681). Clover (at% 15N = 0.3669) was significantly lower than radish shoot (at% 15N = 0.3700) and radish root (at% 15N = 0.3696). Rye (at% 15N = 0.3682) was significantly lower than radish shoot. For the fall, late-planted cover crops, there were significant effects for site (p = 0.0010) and tissue type (p = 0.0407). Site four (at% 15N = 0.3699) was significantly higher than site three (at% 15N = 0.3678). Clover (at% 15N = 0.3675) was significantly lower than radish shoot (at% 15N = 0.3701). For the spring, early-planted cover crops, there was a significant effect for tissue type (at% 15N = 0.0236), but not site. Clover (at% 15N = 0.3666) was significantly lower than rye (at% 15N = 0.3686). For the spring, late-planted cover crops, there was no significant effect for tissue type or site. Because there were differences between sites and/or among tissue types for the early-planted fall samples, late-planted fall samples, and early-planted 102 spring samples, the background at% 15N values for each site x tissue type was analyzed separately. For each site x tissue type, an ANOVA was run to test for differences in the background at% 15N values among tissue types within different cover crops (e.g., radish shoot in radish cover crop versus radish shoot in three-way mix cover crop). For fall and spring, for each site x tissue type, there were no significant effects of tissue types within different cover crops, and therefore the background level at% 15N values were pooled over this factor. The at% 15N of a tissue type in an enriched plot was compared to the at% 15N of the same site, tissue type, and planting date in an unenriched plot (background level). A one-sample t-test was performed to determine if an experimental treatment value of at% 15N was significantly higher than the background level. Because we were only interested in knowing if cover crops from experimental treatments had a higher at% 15N than the background level (not lower), we used a “lower one-sided t-test”, with the null value (H0) being the at% 15N of the experimental plot in question. The null hypothesis is that the mean of control values is equal to the H0; the alternative hypothesis is that the mean of control values is less than the H0. Levels of at% 15N significantly above the background level (control) at% 15N were interpreted to mean that the cover crop captured some of the buried 15N tracer. We evaluated how much 15N buried tracer the cover crops captured. Due to the incomplete factorial design (no two-way mix cover crop planted late, and no 15N buried at 180 cm for the late-planted cover crops), the results from the early-planted cover crops and late-planted cover crops were analyzed separately. An ANOVA was performed to determine if the 15N percent recovery was affected by the fixed effects of 1) cover crop, 103 2) 15N burial depth, 3) species x 15N burial depth, and 4-6) the interactions of all variables above x site. The 15N percent recovery data was not normally distributed so it was log10 transformed, which normalized the distribution. Thus, the dependent variable in the ANOVA was log10 15N percent recovery. To test for differences of 15N percent recovery between cover crop planting dates, an ANOVA was performed including the independent variables of cover crop, cover crop planting date, 15N burial depth, all possible interactions, and the interaction of site with all of the above factors. Experimental units in the three-way mix cover crop and 180 cm 15N burial depth were not included in the analysis since they were not represented in the late planting date treatment. The dependent variable was log10 15N percent recovery. To test if rye from spring sampling had higher 15N percent recovery than rye from fall sampling, an ANOVA was performed including the independent variables of cover crop sampling date and the interaction of sampling date x site. A separate analysis was performed for early-planted cover crops with 15N buried at 60 cm, early-planted cover crops with 15N buried at 120 cm, early-planted cover crops with 15N buried at 180 cm, late-planted cover crops with 15N buried at 60 cm, and late-planted cover crops with 15N buried at 120 cm. The dependent variable was log10 15N percent recovery. We evaluating if corn from experimental treatments contained higher at% 15N than background level. A one-sample t-test was performed to determine if 1) corn V5 and 2) corn grain samples had significantly higher at% 15N than the background level. We analyzed corn V5 and corn grain for the presence of at% 15N in plots from all early- planted cover crop treatments and all no cover crop control treatments. 104 We evaluated how much 15N buried tracer the corn captured. An ANOVA was performed for the experimental units with the 15N buried at 60 cm, to test if the corn V5 and corn grain 15N percent recovery was affected by the fixed effects of cover crop and the interaction of site x cover crop. A separate ANOVA was performed for the experimental units in the no cover crop control treatment, to test if the corn V5 and corn grain 15N percent recovery was affected by the fixed effects of 15N burial depth and the interaction of site x 15N burial depth. Results Experiment #1 Presence of buried 15N tracer in cover crops The at% 15N was significantly higher than the background level, at p < 0.001, in almost every treatment combination (67 out of 72 plots). Exceptions included two plots in which at% 15N was significantly higher than the background level at p < 0.1, specifically, 1.) site two, rep two, late-planted radish root from 100 cm burial, and 2.) site 2, rep 2, early-planted, radish root from 200 cm burial, and three plots in which at% 15N was not significantly higher than the background level, specifically, 1.) site two, rep three, early- planted rye from 200 cm burial, 2.) site one, rep one, early-planted rye from 200 cm burial, and 3.) site one, rep two, late-planted, radish root from 100 cm burial. Percent recovery of buried 15N in cover crops The mean 15N percent recovery of the cover crops ranged from 0.0076% - 35.8%. Table 16 in Appendix 6 list mean, standard deviation, minimum and maximum 15N percent recovery for every treatment combination. There was a significant difference in 105 the log10 15N percent recovery among site x cover crop x planting date (p = 0.0594) and among planting date x 15N burial depth (p < 0.0001) (Table 11). Radish versus rye cover crop species did not have different 15N percent recovery regardless of planting date and site. Across cover crops, an early-planting date resulted in higher 15N recovery from 100 cm deep than from 200 cm deep (p < 0.0001). However, a late-planting date did not result in higher 15N recovery from 100 cm than from 200 cm (p = 0.9714). An early-planting date resulted in higher 15N recovery from 100 cm than a late-planting date (p < 0.0001). However, an early-planting date did not result in higher 15N recovery from 200 cm than a late-planting date (p = 0.2077). The early-planted rye had higher 15N percent recovery than late-planted rye on site one (p = 0.0008) and site two (p = 0.0087). The early-planted radish had higher 15N percent recovery than late-planted radish on site one (p < 0.0001), but not site two (p = 0.7402). The early-planted radish had higher 15N percent recovery on site one than on site two (p = 0.0265). However, the late-planted radish and the early- and late-planted rye did not have differences in 15N percent recovery between sites. Soil sampling at 15N burial points The soil at% 15N in the early-planted rye treatment plots that was buried at 100 cm appeared to move down the soil profile by December and May (Figure 11). Experiment #2 Presence of buried 15N tracer in cover crops In December, the at% 15N was significantly (p < 0.01) higher than the background level for the radish shoot and root in 29 out of 30 plots and for rye in 30 out of 30 plots, regardless of planting-date or 15N burial depth. In May, the rye at% 15N was still significantly (p < 0.01) higher than the background level in 30 of 30 plots (Table 12). 106 In December, within the two-species cover crop, the at% 15N in radish shoot, radish root, and rye always was significantly (p < 0.01) higher than the background where the 15N was buried at 60 cm and in all but one plot where the 15N was buried at 120 cm. Where the 15N was buried at 180 cm, the at% 15N in the radish and rye components of the cover crop were significantly (p < 0.05) higher than the background in five of six plots. By May, within the two-species cover crop, the rye had at% 15N values significantly (p < 0.01) higher than the background in all six of the 60 cm 15N burial plots, in five of the six 120 cm 15N burial plots, and in two of the six 180 cm 15N burial plots (Table 12). By December, within the three-species cover crop, the radish shoot, radish root, rye, and clover (in all but one case) had at% 15N values significantly (p < 0.01) higher than the background where the 15N was buried at 60 cm. Where the 15N was buried at 120 cm, the at% 15N in the radish shoot, radish root, and rye was significantly (p < 0.05) higher than the background in every plot, while the clover had at% 15N values significantly (p < 0.05) higher than the background in six out of 12 plots. When the 15N was buried at 180 cm, the radish shoot, radish root, and rye had at% 15N significantly (p < 0.01) higher than the background in five out of six plots, while the clover had at% 15N values significantly (p < 0.01) higher than the background in three out of six plots. By May, within the three-species cover crop, the rye and clover had at% 15N values significantly (p < 0.01) higher than the background in every plot where the 15N was buried at 60 cm. In the 120 cm 15N burial plots, rye had at% 15N values significantly (p < 0.05) higher than the background in 10 out of 12 plots, while the clover had at% 15N values significantly (p < 0.05) higher than the background in eight out of 11 plots. In the 180 cm 15N burial plots, rye had at% 15N significantly (p < 0.1) higher than the 107 background in four out of six plots, while the clover had at% 15N significantly (p < 0.01) higher than the background in three out of six plots (Table 12). Percent recovery of buried 15N in cover crops By December, within the early-planted cover crop treatment, there were significant (p < 0.0001) differences in the 15N percent recovery among 15N burial depths, with 60 cm burial > 120 cm burial > 180 cm burial. There was no difference among cover crops (p = 0.9897). There was no difference in the 15N percent recovery between two-way mix and three-way mix cover crops for the 60 cm 15N burial depth (p = 1.0000), 120 cm 15N burial depth (p = 1.0000), or 180 cm 15N burial depth (p = 1.0000). Within the late- planted cover crop treatment, there were significant (p < 0.0001) differences in the 15N percent recovery between 15N burial depth treatments, with 60 cm burial > 120 cm. There was no difference among cover crops (p = 0.308). By December, the early-planted cover crops had higher 15N percent recovery than the late-planted cover crops (p < 0.0001). For fall growth, early-planted cover crops captured on average 14.5% of the buried 15N from 60 cm, 2.67% of the buried 15N from 120 cm, and 0.31% of the buried 15N from 180 cm. Late-planted cover crops captured on average 1.36% of the buried 15N from 60 cm and 0.07% of the buried 15N from 120 cm (Figure 12). Table 16 and Table 17 in Appendix 6 list the mean, standard deviation, minimum and maximum 15N percent recovery for every treatment combination. Cover crop sampling season differences We assessed the rye 15N percent recovery for the December sampling versus the May sampling. For the early-planted rye, the percent recovery was higher in the fall than the spring for 60 cm, 120 cm, and 180 cm burial depths. For the late-planted rye, the 108 spring percent recovery (6.58%) was higher than the fall (1.45%) for the 60 cm burial, although not significantly (p = 0.1042), and the spring percent recovery (0.31%) was significantly (p = 0.0318) higher than the fall (0.09%) for the 120 cm burial, although by a very small amount (Table 13). Presence of buried 15N in corn In plots where no cover crop was planted, V5 corn had at% 15N significantly (p < 0.1) above background (no 15N application) level in four out of six replications when 15N was buried at 60 cm, and three out of six replications when 15N was buried at 120 cm and 180 cm. In plots where no cover crop was planted, corn grain had at% 15N significantly (p < 0.05) above background (no 15N application) level in four out of six replications when 15N was buried at 60 cm, four out of six replications when 15N was buried at 120 cm, and one out of six replications when 15N was buried at 180 cm. In plots that had cover crops, V5 corn and corn grain always had at% 15N significantly (p < 0.1) above background (no 15N application) level, regardless of the cover crop species (Table 14). In plots were no cover crop was planted, the 15N percent recovery of V5 corn and corn grain was not different among 15N burial depths. For the 60 cm 15N burial depth, there were differences in V5 corn (but not corn grain) 15N percent recovery among previous cover crop treatments. Because there was a significant (p = 0.0064) cover crop by site interaction, sites were analyzed separately. The distribution of the data was less skewed when sites were analyzed separately. At site four, the 15N percent recovery of the V5 corn in the three-way mix treatment (0.399%) was greater than the V5 corn in the no cover crop control treatment (0.064%) (p = 0.0885). At site three, the 15N percent recovery of the V5 corn in the three-way mix treatment (0.0989%) was greater than the 109 V5 corn in the no cover crop control treatment (0. 0061%) (p = 0.0945). Although due to the design of the experiment and the sampling protocols these are all under estimates, in all cases, the amount of N that was traced through the V5 corn was less than 6% of the N taken up by the three-way mix cover crop. Soil sampling at 15N burial points Figure 13 depicts soil at% 15N in soil cores taken from plots that had no cover crop and 15N was buried at 60 cm deep. Soil cores were taken in February (site three) or April (site four) 2016, June 2016, and October 2016. Figure 14 depicts soil at% 15N in soil cores taken in February (site three) or April (site four) 2016 from plots that had no cover crop and 15N was buried at 120 cm deep. Discussion All species of cover crops, both early- and late-planted, contained levels of 15N higher than the background level, regardless of whether it was buried at 60 cm, 120 cm, or 180 cm. A few plots did not contain enriched 15N; however, these exception plots were not consistent between replications in a treatment. This finding was contrary to our hypothesizes, as we expected late-planted cover crops not to capture 15N from 120 cm or 180 cm. Levels of at% 15N above the background level indicate that the tissue contained 15N that originated from the buried tracer, either through the plant scavenging the tracer or the tracer being transferred between plants within a plot. The comparisons to see if the at% 15N is above the background are qualitative comparisons, not quantitative comparisons. 110 We performed quantitative comparisons between cover crop treatments by comparing cover crop 15N percent recovery. The 15N percent recovery values will be underestimates of the actual percent recover, as we had an unconfined system, and assumed there would be no 15N in plants greater than 20 cm from the burial point. Furthermore, we did not account for any 15N from the rye roots, clover roots, or radish fine roots. While we could not estimate the total plant recovery, the 15N percent recovery values that we did estimate allowed us to make relative comparisons between cover crops and planting dates, which was the main goal of the study. In experiment #1, early-planted cover crops recovered on average 13.7% of the buried 15N in the 100 cm burial plots, 52 times more than the late-planted cover crops, which only recovered 0.26% of the buried 15N in the 100 cm burial plots. In experiment #2, the early-planted cover crops took up on average 14.5% of the 15N that was buried at 60 cm, while the late-planted cover crops took up only 1.4%. In other words, the early- planted cover crops took up 10 times more 15N than the late-planted cover crops from 60 cm deep. While the percent recovery of the buried 15N from 120 cm deep was small in all cases, the early-planted cover crops took up 38 times more 15N (2.67%) than the late- planted cover crops (0.07%) from 120 cm deep. While the 15N percent recovery amounts from the 120+ cm burial depth were small, these findings support that radish and rye species both seem capable of quick, deep, root growth and are therefore promising “catch-crops”. In a study at Rothamsted and at Woburn Experimental Farms in Bedfordshire that investigated winter wheat planting dates, Barraclough and Leigh (1984) found that for the September planting, roots were present 1 m deep by December, but for the October planting, roots did not reach 1 m until April. Furthermore, they found that 111 September-planted wheat had over four times as much root dry weight and root length by March than October-planted wheat (Barraclough and Leigh, 1984). We expected that clover within the three-way mix would not be able to scavenge 15N from the deeper depths (120 cm, 180 cm). However, when 15N was buried at 120 cm, 15N was found in the clover tissue of 33% of the early-planted three-way mix replications and 67% of the late-planted three-way mix replications, and when 15N was buried at 180 cm, 15N was found in the clover tissue of 50% of the early-planted three-way mix replications. Because we did not have a monoculture clover treatment, we cannot know for certain if the clover was actually able to reach the deep buried 15N. However, we believe it is more likely that the clover picked up 15N from its deeper rooted neighbors (radish and rye), through sloughed cells, root turnover, or N leaching from senescing leaves (Dabney, et al., 2010; Maeght, et al., 2013; Smil, 1999). We found no evidence that adding clover to the mix hindered the N uptake of radish or rye, a concern of some practitioners and policy makers. Both the radish and rye cover crops contained tracer from 180 cm deep regardless of whether they were part of a mixed stand with clover or not. Furthermore, there was no difference in the amount of 15N taken up by the two-way mix versus the three-way mix cover crops when 15N was buried at 60 cm, 120 cm, or 180 cm. Tosti and Thorup-Kristensen (2010) also found in a study using a mix of plant species with different colored roots found that the maximum root depth and depth penetration rate of beet was not affected by the presence of legumes. There is a risk that if a cover crop does not scavenge deep soil NO3 in the fall, the NO3 would be out of reach for the spring cover crop. This is especially true in some environments with sandier soil or more precipitation, where NO3 may leach rapidly 112 through the soil profile. In such environments, cover crops with fast growing roots may be the optimal system to capture NO3 before it has a chance to leach to soil depths below the root zone. When we compared the percent recovery of the buried 15N in the fall rye growth versus spring growth, we found that the late-planted rye does take up additional 15N during spring growth, but not amounts that would be expected to have an impact on reducing NO3 leaching or supplying N to a following crop. We found evidence that corn following a cover crop was more likely to be enriched in 15N or have greater uptake of 15N than corn that did not follow a cover crop. However, the percent recoveries that we observed were very low (< 1%), regardless of whether there was a previous cover crop or not. Our study did not provide evidence that cover crops will recycle meaningful amounts of N from deep soil layers (60+ cm) to the subsequent corn. However, we believe this is a result of limitations of our experimental design. Specifically, we had an unconfined system, and therefore 15N may have moved horizontally outside of our sampling circle. We also did not account for 15N in the corn roots at the V5 sampling time, and we did not account for 15N in the corn roots or corn plant at the corn grain sampling time. Forage radish could provide N to the following corn crop, as it almost always winter-kills in Maryland and quickly decomposes, releasing mineral N into the soil surface layers (0-60cm) (Dean and Weil, 2009), although some studies have found that forage radish offers no N fertilizer replacement value (Ruark, et al., 2018). Crimson clover has been found to increase the following corn yield and corn N content (Kramberger, et al., 2009). Other studies provide evidence that mixed species or legume cover crops can increase subsequent corn yield (Marcillo and Miguez, 2017). 113 Conclusions and practical applications Cover crops perform best if planted as early as possible. Our data show that early- September planting will allow cover crops to capture substantially more N than early- October planted cover crops. Thus, management practices such as shifting to earlier maturing corn hybrids and relay-crop establishment of cover crops should be evaluated. If planted by early-September, cover crops of radish, rye, and mixes of radish and rye (with or without crimson clover) were all effective at scavenging deep soil N. We found evidence that a late-planted rye cover crop will be able to scavenge additional N in the spring. Therefore, we recommend that rye cover crops be allowed to grow as much as possible in the spring to continue scavenging N. However, our findings from the 15N tracer in the soil profiles provide evidence that in some cases, nitrate is leaching from late-summer to spring, and therefore, rye will need to catch-up with the leaching N. 114 Table 7. Experiment #1, soil pH, percent sand, percent clay, percent C, percent N, NH4-N (kg N ha -1), and NO3-N (kg N ha -1) for each 20 cm soil depth increment (0-200 cm) and P, K, Mg, Ca, and S (mg kg-1) for 0-30 cm soil. Reported pH, sand, clay, C, N, P, K, Mg, Ca, and S values are the average from three soil cores, one per block. Reported NO3-N and NH4-N values are the average from six soil cores, two per block. Soil depth pH Sand Clay Soil texture C N NH4-N NO3-N P K Mg Ca S -1cm % % kg N ha-1 mg kg 0-20 5.52 85.4 2.9 Loamy fine sand 0.649 0.0340 20.5 35.3 95.3 49.0 37.3 269 8.00 20-40 5.87 85.3 3.3 Loamy fine sand 0.288 BDL 9.14 21.6 56.0 43.7 29.0 203 4.67 40-60 5.99 83.4 3.9 Loamy fine sand 0.143 BDL 5.49 13.7 -- -- -- -- -- 60-80 6.13 85.4 4.3 Loamy fine sand 0.126 BDL 7.35 13.6 -- -- -- -- -- 80-100 6.35 82.8 6.1 Loamy fine sand 0.0846 BDL 3.85 6.15 -- -- -- -- -- Site one 100-120 6.31 76.9 5.3 Loamy fine sand 0.0748 BDL 3.40 4.22 -- -- -- -- -- 120-140 6.13 74.3 6.8 Sandy loam 0.0667 BDL 3.23 4.15 -- -- -- -- -- 140-160 5.84 72.7 8.6 Sandy loam 0.0569 BDL 5.25 5.34 -- -- -- -- -- 160-180 5.85 70.3 9.6 Sandy loam 0.0709 BDL 3.59 4.25 -- -- -- -- -- 180-200 5.31 70.8 13.1 Sandy loam 0.0524 BDL 4.47 4.81 -- -- -- -- -- 0-20 4.79 26.0 27.6 Clay loam 1.50 0.109 34.4 49.5 16.3 77.7 56.3 402 28.3 20-40 4.71 20.0 37.1 Silty clay loam 0.780 0.0597 18.8 14.0 5.0 49.3 49.3 377 53.0 40-60 4.27 12.1 53.5 Clay 0.345 0.0350 12.0 6.39 -- -- -- -- -- 60-80 4.05 10.2 56.7 Clay 0.231 0.0297 10.3 5.13 -- -- -- -- -- 80-100 4.07 10.2 53.5 Clay 0.221 0.0247 8.87 4.40 -- -- -- -- -- Site two 100-120 3.98 10.6 54.5 Clay 0.205 0.0273 8.96 4.13 -- -- -- -- -- 120-140 3.99 10.4 53.5 Clay 0.171 BDL 7.77 3.34 -- -- -- -- -- 140-160 3.98 12.9 47.2 Clay 0.157 BDL 7.13 3.05 -- -- -- -- -- 160-180 4.01 12.6 46.4 Silty clay 0.149 BDL 6.53 2.79 -- -- -- -- -- 180-200 4.02 11.0 51.0 Clay 0.158 BDL 6.86 2.95 -- -- -- -- -- 115 Table 8. Experiment #2, soil pH, percent sand, percent clay, percent C, percent N, NH4-N (kg N ha -1), and NO -13-N (kg N ha ) for each 15 cm soil depth increment (0-210 cm) and P, K, Mg, Ca, and S (mg kg-1) for top three 15 cm soil depth increments (0-45 cm). Reported pH, sand, clay, C, N, P, K, Mg, Ca, and S values are the average from six cores (two cores 10 cm apart composited, taken in each of three blocks). Reported NO3-N and NH4-N values are the average from 15 cores (five cores in each of three blocks). pH Sand Clay Soil texture C N NH4-N NO3-N P K Mg Ca S Soil depth % % kg N ha-1 mg kg-1 0-15 6.14 77.0 3.4 Loamy fine sand 0.654 0.054 60.0 42.0 52.7 382 3.67 15.2 43.1 15-30 6.29 74.6 5.5 Sandy loam 0.275 0.023 48.0 34.7 42.7 311 3.00 30-45 6.48 65.6 8.2 Sandy loam 0.158 BDL 23.0 38.3 50.0 287 2.33 8.65 8.68 45-60 6.52 65.6 9.5 Sandy loam 0.126 BDL -- -- -- -- -- 60-75 6.38 69.3 8.5 Sandy loam 0.103 BDL -- -- -- -- -- 9.13 12.2 75-90 6.38 75.0 6.1 Sandy loam 0.053 BDL -- -- -- -- -- 90-105 6.32 79.1 6.9 Loamy fine sand 0.050 BDL -- -- -- -- -- Site three 7.68 3.57 105-120 5.95 75.6 9.7 Sandy loam 0.051 BDL -- -- -- -- -- 120-135 5.99 63.8 13.2 Sandy loam 0.098 BDL -- -- -- -- -- 8.65 8.54 135-150 5.85 68.5 12.5 Sandy loam 0.067 BDL -- -- -- -- -- 150-165 5.84 63.6 13.0 Sandy loam 0.038 BDL -- -- -- -- -- 9.58 3.95 165-180 5.46 57.5 14.7 Sandy loam 0.036 BDL -- -- -- -- -- 180-195 5.39 51.2 16.5 Loam 0.038 BDL -- -- -- -- -- -- -- 195-210 4.83 47.7 20.2 Loam 0.044 BDL -- -- -- -- -- 0-15 6.05 64.8 6.7 Sandy loam 0.883 0.071 36.6 55.1 119.7 38.0 80.7 569 8.00 15-30 6.09 61.4 8.9 Sandy loam 0.368 0.032 56.3 26.3 44.7 389 4.67 30-45 6.05 58.2 12.1 Sandy loam 0.154 BDL 24.5 10.4 4.0 27.3 35.0 365 14.7 Site four 45-60 5.50 55.3 15.9 Sandy loam 0.103 BDL -- -- -- -- -- 60-75 5.06 47.4 18.0 Loam 0.134 BDL 24.7 8.99 -- -- -- -- -- 75-90 4.75 41.1 18.0 Loam 0.074 BDL -- -- -- -- -- 90-105 4.34 39.9 20.8 Loam 0.083 BDL 24.5 12.2 -- -- -- -- -- 116 105-120 4.20 27.7 21.9 Silt loam 0.073 BDL -- -- -- -- -- 120-135 4.23 28.2 21.5 Silt loam 0.095 BDL 28.4 16.0 -- -- -- -- -- 135-150 4.17 26.1 19.8 Silt loam 0.068 BDL -- -- -- -- -- 150-165 4.20 25.9 18.3 Silt loam 0.058 BDL 28.0 14.9 -- -- -- -- -- 165-180 4.07 25.9 17.8 Silt loam 0.064 BDL -- -- -- -- -- 180-195 4.12 34.3 18.0 Loam 0.066 BDL -- -- -- -- -- -- -- 195-210 4.07 27.9 16.5 Silt loam 0.058 BDL -- -- -- -- -- 117 Table 9. Experiment #2, experimental treatment combinations. Experimental factors that defined the treatments included cover crop, cover crop planting date, and 15N burial depth. The cover corps indicated in white were only planted early, not late. The cover crops indicated in grey were planted early and late. Cover crop 15N burial depth Radish 60 cm Radish 120 cm Radish 180 cm Radish No 15N Rye 60 cm Rye 120 cm Rye 180 cm Rye No 15N Two-way mix 60 cm Two-way mix 120 cm Two-way mix 180 cm Two-way mix No 15N Three-way mix 60 cm Three-way mix 120 cm Three-way mix 180 cm Three-way mix No 15N No cover crop 60 cm No cover crop 120 cm No cover crop 180 cm No cover crop No 15N 118 Table 10. Experiment #2, soil samples taken (and depths analyzed in parentheses) per block. The number of asterisks indicate the number of composite cores per sample. Two cores from the same distance from the tracer (dist 15N) were from two different burial points within a plot; two cores from different dist 15N were from one burial point within the plot. 60 cm 15N depth 120 cm 15N depth 10 cm dist 15N 20 cm dist 15N 10 cm dist 15N 20 cm dist 15 N Feb (site three)/ * * * * Apr (site four) (30-120 cm) (30-120 cm) (60-210 cm) ** ** - - Jun (45-210 cm) (45-210 cm) ** - - - Oct (45-210 cm) 119 Table 11. Experiment #1, analysis of variance (ANOVA) tests of fixed effects for log10 15N percent recovery Experimental factor p-value Cover crop 0.044 Planting date 0.015 15N burial depth <0.0001 Cover crop x Planting date 0.57 Planting date x 15N burial <0.0001 Cover crop x 15N burial 0.97 Cover crop x Planting date x 15N burial 0.76 Site x Planting date 0.013 Site x Cover crop 0.16 Site x 15N burial 0.56 Site x Cover crop x Planting date 0.058 Site x Planting date x 15N burial 0.099 Site x Cover crop x 15N burial 0.88 Site x Planting date x Cover crop x 15N burial 0.49 120 Table 12. Experiment #2, percent of the six replications within a given treatment with cover crop tissue type at% 15N significantly (p < 0.01) above background (no 15N application) level. radish radish Planting date rye shoot clover shoot cover crop shoot root fall fall fall spring fall spring 15 60 cm N burial Replications with at% 15N above background (%) Early-planted 100 100 -- -- -- -- Radish Late-planted 100 100 -- -- -- -- Early-planted -- -- 100 100 -- -- Rye Late-planted -- -- 100 100 -- -- Two-way Early-planted mix 100 100 100 100 -- -- Three-way Early-planted 100 100 100 100 100 100 mix Late-planted 100 100 100 100 83 100 120 cm 15N burial Replications with at% 15N above background (%) Early-planted 100 100 -- -- -- -- Radish Late-planted 83 83 -- -- -- -- Early-planted -- -- 100 100 -- -- Rye Late-planted -- -- 100 100 -- -- Two-way Early-planted 83 100 100 83 mix -- -- Three-way Early-planted 100 100 100 67 33 100 mix Late-planted 100* 100 100 100* 67* 50* 15 180 cm N burial Replications with at% 15N above background (%) Early-planted 100 100 -- -- -- -- Radish Late-planted -- -- -- -- -- -- Early-planted -- -- 100 100 -- -- Rye Late-planted -- -- -- -- -- -- Two-way Early-planted 67† 83* 83 33 mix -- -- † Three-way Early-planted 83 83 83 67 50 50 mix Late-planted -- -- -- -- -- -- * p < 0.05 † p < 0.1 121 Table 13. Experiment #2, 15N percent recovery for fall and spring rye. The p-values indicated differences between fall and spring log10 15N percent recovery. 15N burial Fall 15N percent Spring 15N percent p-value Cover crop planting date depth recovery recovery 60 cm 16.07% 6.94% 0.30 Early-planting 120 cm 1.22% 0.78% 0.11 180 cm 0.19% 0.10% 0.036 60 cm 1.45% 6.58% 0.10 Late-planting 120 cm 0.09% 0.31% 0.032 122 Table 14. Experiment #2, percent of the six replications within a given treatment with V5 corn or corn grain at% 15N significantly (p < 0.01) above background (no 15N application) level. Previous cover crop V5 corn Corn grain 60 cm 15N burial Early-planted, radish 100 100 Early-planted, rye 100* 100 Early-planted, two-way mix 100† 100 Early-planted, three-way mix 100 100 No cover crop 67† 67 120 cm 15N burial No cover crop 50† 67 180 cm 15N burial No cover crop 50† 17* * p < 0.05 † p < 0.1 123 30 25 25 20 20 15 15 10 10 5 5 0 0 Precipitation (mm) Temperature (°C) Figure 7. Experiment #1, temperature (°C) and precipitation (mm) from 1 Sep 2014 to 30 Nov 2014. 124 Temperature (°C) 9/1/2014 9/6/2014 9/11/2014 9/16/2014 9/21/2014 9/26/2014 10/1/2014 10/6/2014 10/11/2014 10/16/2014 10/21/2014 10/26/2014 10/31/2014 11/5/2014 11/10/2014 11/15/2014 11/20/2014 11/25/2014 11/30/2014 Precipitation (mm) Early-planted Late-planted 15 15 15 15 100 cm N 200 cm N No burial 100 cm N 200 cm N No burial Rep 1 15 15 15 15 200 cm N No burial 100 cm N 200 cm N 100 cm N No burial 15 15 15 15 200 cm N 100 cm N No burial 200 cm N 100 cm N No burial Site one Rep 2 15 200 cm N No burial 15 15 15 100 cm N 200 cm N No burial 100 cm N 15 No burial 15 15 15100 cm N 200 cm N 200 cm N 100 cm N No burial Rep 3 15 15 15 15 200 cm N No burial 100 cm N 100 cm N 200 cm N No burial Early-planted Late-planted 15 15 200 cm N No burial 100 cm N No burial 15 15 100 cm N 200 cm N Rep 4 15 100 cm N No burial 15 15 200 cm N 200 cm N No burial 15 100 cm N No burial 15 15 15 15100 cm N 200 cm N 200 cm N 100 cm N No burial Site two Rep 5 15 200 cm N No burial 15 15 15 100 cm N 100 cm N 200 cm N No burial 15 No burial 15 15 15100 cm N 200 cm N 200 cm N 100 cm N No burial Rep 6 No burial 15 15 15 15200 cm N 100 cm N 200 cm N No burial 100 cm N Rye Radish Figure 8. Experiment #1, split-split plot experimental design and treatments, showing cover crop planting date as the main plot factor, cover crop as the split-plot factor, and 15N burial depth as the split-split plot factor. 125 25 80 15 60 5 40 -5 20 -15 0 Precipitation (mm) Temperature (°C) Figure 9. Experiment #2, temperature (°C) and precipitation (mm) from 3 Sep 2015 to 7 Oct 2016. 126 Temperature (°C) 1-Sep-15 1-Oct-15 1-Nov-15 1-Dec-15 1-Jan-16 1-Feb-16 1-Mar-16 1-Apr-16 1-May-16 1-Jun-16 1-Jul-16 1-Aug-16 1-Sep-16 1-Oct-16 Precipitation (mm) Figure 10. Experiment #2, horizontal spatial arrangement of 15N burial holes (red dots) and areas around burial points in which biomass was sampled (green circles). 127 Figure 11. Experiment #1, soil at% 15N in soil cores taken in December 2014 (site one) and May 2015 (sites one and two) from early-planted rye plots in which 15N was buried at 100 cm. The at% 15N values are the average of two blocks in December and three blocks in May. 128 16% a 14% 12% 10% 8% 6% 4% b 2% a c b 0% 60 cm 120 cm 180 cm 60 cm 120 cm Early-planted Late-planted Figure 12. Experiment #2, December 15N percent recovery from each 15N burial depth for early- and late-planted cover crops (across all cover crops). The 15N burial depth values for log10 15N percent recovery within the same cover crop planting date treatment followed by the different letters are significantly different (p < 0.05). 129 15N percent recovery Figure 13. Experiment #2, soil at% 15N in soil cores taken in February 2016 (site three) or April 2016 (site four), June 2016 (sites three and four), and October 2016 (sites three and four) from the no cover crop control plots in which 15N was buried at 60 cm. The at% 15N values are the average of three blocks. 130 Figure 14. Experiment #2, soil at% 15N in soil cores taken in February 2016 (site three) or April 2016 (site four) from the no cover crop control plots in which 15N was buried at 120 cm. The at% 15N values are the average of three blocks. 131 Appendix 5. Detailed methods for estimation of rye biomass Table 15. Samples taken to estimate rye biomass, the number of samples (N) taken per treatment, regression equations relating rye patchiness, height, and/or percent cover to biomass, and adjusted R2 for each regression equation Sample Treatment Samples taken N Equation Adj date R2 Fall rye At site 3 and site 4, five samples were taken 20 Dry matter = 0.91078*(height) – 0.95 in early-planting plots, and five in late- 0.47720*(patchiness) + planting plots 0.24967*(percent cover) 7 Jan 2016 Fall two-way mix At site 3 and site 4, five samples were taken 20 dry matter = 0.42022*(percent cover1) 0.91 and three-way mix in early-planting plots, and five in late- planting plots Spring rye At site 3 and site 4, three samples were taken 12 dry matter = 0.29445*(height) + 0.87 in early-planting plots, and three in late- 1.40593*(percent cover) planting plots 8 May Spring two-way At site 3 and site 4, four samples were taken 28 dry matter = 0.04924*(height) + 0.89 2016 mix and three-way in two-way mix plots, five in early-planting 0.66463*(patchiness) + mix three-way mix, and five in late-planting 1.07426*(percent cover) three-way mix 1 We used only the percent cover independent variable for the fall rye in mixed cover crop treatments. Fall samples were not taken in two-way mix or three- way mix treatments; therefore we needed to use fall samples from rye treatments. Rye growth within rye treatment was shorter than rye growth in mixed treatments, likely because rye in mixed treatments was growing tall to compete for sunlight with the radish. Therefore, using the rye height variable resulted in what visually appeared to be unrealistically high values for biomass. Including the variable of patchiness also resulted in what visually appeared to be unrealistically high values for biomass. Rye percent cover alone resulted in what visually appeared to be realistic estimates for biomass. 132 Appendix 6. 15N percent recovery data Table 16. Percent recovery of 15N for December sampled radish, rye, two-way mix (2mix), and three-way mix (3mix) cover crops, showing the number of observations per reported value (N), mean, standard deviation (SD), minimum value (Min) and maximum value (Max) Exp. # Site Planting date Cover crop N Mean SD Min Max 100 cm 15N burial Exp. #1 Site 2 Early Radish 3 4.68% 0.0622 0.55% 11.8% Exp. #1 Site 1 Early Radish 3 35.8% 0.158 22.3% 53.2% Exp. #1 Site 2 Early Rye 3 3.32% 0.0273 0.46% 5.89% Exp. #1 Site 1 Early Rye 3 10.9% 0.109 0.50% 22.3% Exp. #1 Site 2 Late Radish 3 0.97% 0.016172 0.01% 2.84% Exp. #1 Site 1 Late Radish 3 0.03% 0.000093 0.02% 0.04% Exp. #1 Site 2 Late Rye 3 0.05% 0.000605 0.01% 0.12% Exp. #1 Site 1 Late Rye 3 0.01% 0.000029 0.00% 0.01% 200 cm 15N burial Exp. #1 Site 2 Early Radish 3 0.05% 0.000234 0.03% 0.07% Exp. #1 Site 1 Early Radish 3 0.64% 0.00460 0.34% 1.17% Exp. #1 Site 2 Early Rye 3 0.10% 0.00141 0.00% 0.26% Exp. #1 Site 1 Early Rye 3 0.07% 0.000525 0.01% 0.11% Exp. #1 Site 2 Late Radish 3 0.09% 0.000404 0.05% 0.13% Exp. #1 Site 1 Late Radish 3 0.07% 0.000174 0.05% 0.08% Exp. #1 Site 2 Late Rye 3 0.02% 0.000260 0.01% 0.05% Exp. #1 Site 1 Late Rye 3 0.03% 0.000196 0.01% 0.05% 60 cm 15N burial Exp. #2 Site 4 Early 2mix 3 18.1% 0.0444 13.0% 21.4% Exp. #2 Site 3 Early 2mix 3 10.6% 0.0352 7.27% 14.3% Exp. #2 Site 4 Early 3mix 3 15.7% 0.0452 12.2% 20.8% Exp. #2 Site 3 Early 3mix 3 7.68% 0.0435 3.16% 11.8% Exp. #2 Site 4 Early Radish 3 19.0% 0.0391 14.5% 21.4% Exp. #2 Site 3 Early Radish 3 12.4% 0.130 1.53% 26.9% Exp. #2 Site 4 Early Rye 3 26.3% 0.211 2.98% 43.9% Exp. #2 Site 3 Early Rye 3 5.81% 0.0572 2.06% 12.4% Exp. #2 Site 4 Late 3mix 3 0.60% 0.00230 0.46% 0.87% Exp. #2 Site 3 Late 3mix 3 0.27% 0.00219 0.14% 0.52% Exp. #2 Site 4 Late Radish 3 1.91% 0.0177 0.12% 3.67% Exp. #2 Site 3 Late Radish 3 2.48% 0.0256 0.50% 5.37% Exp. #2 Site 4 Late Rye 3 2.24% 0.0282 0.34% 5.48% 133 Exp. #2 Site 3 Late Rye 3 0.67% 0.00609 0.03% 1.25% 120 cm 15N burial Exp. #2 Site 4 Early 2mix 3 1.53% 0.0108 0.30% 2.26% Exp. #2 Site 3 Early 2mix 3 6.34% 0.106 0.12% 18.5% Exp. #2 Site 4 Early 3mix 3 1.41% 0.00408 0.98% 1.80% Exp. #2 Site 3 Early 3mix 3 5.26% 0.0727 0.16% 13.6% Exp. #2 Site 4 Early Radish 3 0.41% 0.00207 0.28% 0.65% Exp. #2 Site 3 Early Radish 3 3.95% 0.0479 0.03% 9.30% Exp. #2 Site 4 Early Rye 3 1.13% 0.00741 0.43% 1.91% Exp. #2 Site 3 Early Rye 3 1.31% 0.00597 0.68% 1.87% Exp. #2 Site 4 Late 3mix 3 0.07% 0.000257 0.04% 0.08% Exp. #2 Site 3 Late 3mix 3 0.05% 0.000405 0.01% 0.09% Exp. #2 Site 4 Late Radish 3 0.04% 0.000333 0.00% 0.06% Exp. #2 Site 3 Late Radish 3 0.07% 0.000607 0.03% 0.14% Exp. #2 Site 4 Late Rye 3 0.13% 0.000894 0.03% 0.20% Exp. #2 Site 3 Late Rye 3 0.06% 0.000332 0.02% 0.08% 180 cm 15N burial Exp. #2 Site 4 Early 2mix 3 0.12% 0.000479 0.07% 0.16% Exp. #2 Site 3 Early 2mix 3 0.48% 0.00539 0.02% 1.08% Exp. #2 Site 4 Early 3mix 3 0.32% 0.00377 0.01% 0.74% Exp. #2 Site 3 Early 3mix 3 0.22% 0.00226 0.08% 0.48% Exp. #2 Site 4 Early Radish 3 0.29% 0.00229 0.07% 0.53% Exp. #2 Site 3 Early Radish 3 0.65% 0.00491 0.25% 1.20% Exp. #2 Site 4 Early Rye 3 0.24% 0.000795 0.16% 0.32% Exp. #2 Site 3 Early Rye 3 0.15% 0.00102 0.07% 0.26% 134 Table 17. Percent recovery of 15N for April sampled two-way mix (2mix), three-way mix (3mix), and rye cover crops, showing the number of observations per reported value (N), mean, standard deviation (SD), minimum value (Min) and maximum value (max) Site Planting date Cover crop N Mean SD Min Max 60 cm 15N burial Site 4 Early 2mix 3 2.73% 0.0236 0.90% 5.40% Site 3 Early 2mix 3 0.97% 0.00193 0.78% 1.16% Site 4 Early 3mix 3 2.02% 0.0131 0.68% 3.29% Site 3 Early 3mix 3 0.63% 0.00292 0.30% 0.81% Site 4 Early Rye 3 11.2% 0.0222 9.40% 13.7% Site 3 Early Rye 3 2.68% 0.0171 1.65% 4.65% Site 4 Late 3mix 3 5.80% 0.0540 2.50% 12.0% Site 3 Late 3mix 3 0.14% 0.000551 0.08% 0.19% Site 4 Late Rye 3 12.5% 0.104 0.74% 20.5% Site 3 Late Rye 3 0.68% 0.00672 0.05% 1.39% 120 cm 15N burial Site 4 Early 2mix 3 0.11% 0.000798 0.02% 0.16% Site 3 Early 2mix 3 1.20% 0.0196 0.07% 3.46% Site 4 Early 3mix 3 0.16% 0.00197 0.04% 0.38% Site 3 Early 3mix 3 0.62% 0.00874 0.03% 1.62% Site 4 Early Rye 3 0.54% 0.000639 0.48% 0.61% Site 3 Early Rye 3 1.02% 0.00947 0.39% 2.11% Site 4 Late 3mix 3 0.31% 0.00308 -0.04% 0.53% Site 3 Late 3mix 3 0.37% 0.00512 0.04% 0.96% Site 4 Late Rye 3 0.47% 0.00300 0.28% 0.81% Site 3 Late Rye 3 0.15% 0.000499 0.09% 0.19% 180 cm 15N burial Site 4 Early 2mix 3 0.04% 0.000240 0.02% 0.07% Site 3 Early 2mix 3 0.09% 0.00129 0.00% 0.23% Site 4 Early 3mix 3 0.03% 0.000387 0.00% 0.07% Site 3 Early 3mix 3 0.02% 0.000115 0.01% 0.03% Site 4 Early Rye 3 0.12% 0.000559 0.08% 0.19% Site 3 Early Rye 3 0.07% 0.000338 0.05% 0.11% 135 Chapter 4: Cover crop systems influence on deep soil N dynamics and the following corn crop: on-farm investigations Abstract In the Mid-Atlantic USA, substantial mineral N (100-500 kg N ha-1) remains in the 0-2 m soil in September, 78% deeper than 30 cm, which is at risk to leach over winter months. We hypothesized that deep-rooted cover crops planted by early-September could capture residual N, and potentially recycle this N for following cash crops. We performed experiments on 19 farms in Maryland and Pennsylvania investigating the effects of four cover crop systems (forage radish, winter cereal, forage radish + winter cereal + crimson clover, no cover crop control) on cover crop biomass, N uptake, and inorganic N distribution within the upper 210 cm of soil in late-fall and early-spring, and the following corn crop’s growth and yield. In late-fall, radish reduced soil NO3-N to 90 cm deep, while winter cereal or mix cover crops reduced NO3-N to 60 cm deep. In the spring, radish released NO3-N on the soil surface (0-30 cm), but was less effective than winter cereal at reducing NO3 from 30-150 cm deep. Winter cereal was the most effective at reducing soil NO3 throughout the entire soil profile. Mix was more effective than winter cereal and as effective as radish at ensuring available NO3 on the soil surface (0- 30 cm), and was as effective as winter cereal in reducing soil NO3 from 30-210 cm soil. The V5 corn biomass and N content were affected by the previous cover crop treatment in the order radish > mix = control > winter cereal. At the farmers’ standard N fertilizer application rate, corn yield following radish or control was higher than winter cereal, and corn yield following radish was higher than mix. Cover crops can be fit within the 136 framework of existing crop systems to scavenge and accumulate N, and through their decomposition supply N for subsequent crops, therein improving the overall N use efficiency of the cropping system. Introduction Cropping systems, weather patterns, and soil characteristics in the Mid-Atlantic USA make agricultural systems in this region prone to nitrate (NO3) leaching. A common cropping system in Maryland is a corn (Zea mays L.) to soybean (Glycine max (L.) Merr.) rotation. In this rotation, from September to May there is no crop actively taking up nitrogen (N) from the soil. In addition, soybean acquires 50-60% of its N through symbiotic N fixation, and therefore, does not scavenge N from the soil profile as efficiently as non-legume crops (Salvagiotti, et al., 2008). The region has a humid climate with annual rainfall relatively uniformly distributed throughout the entire year. The average annual precipitation for Beltsville, MD is 1063 mm (US Climate Data, 2018). Therefore, in the case of the typical corn/soybean rotation, the majority of the rainfall is occurring when there is no crop growing and taking up N or water from the soil profile. Many soils of the Coastal Plain physiographic region surrounding the Chesapeake Bay have sandy textures, through which NO3 leaches more rapidly than through the finer textured soils of the Piedmont and other areas with soils formed from metamorphic and sedimentary rock. Plants grown in the fall, following harvest of the cash crop, are called cover crops, catch crops, or green manures (Thorup-Kristensen, et al., 2003). Some cover crop species have the potential to quickly grow deep roots, and could serve as a “catch crop” to 137 capture NO3 in the fall months before it leaches out of reach, and potentially release N in the spring months to be used by the following cash crop (Dabney, et al., 2010; Meisinger, et al., 1990; Meisinger, et al., 1991). For example, while corn roots did not reach depths > 1.2 m, subsequent winter wheat could use soil NO3 up to 2 m deep (Zhou, et al., 2008). Cover crops can serve to reduce NO3 concentration in aquifers used for drinking water and to decrease NO3 concentrations in surface waters, lessening the risk of eutrophication and associated negative environmental effects (Thorup-Kristensen, et al., 2003). Cover crops can be fit within the framework of the existing crop system to scavenge and accumulate N in their tissue, and then through their decomposition, to supply N for subsequent crops. Such a cover crop system would improve the overall N use efficiency of the cropping system. To capture N before it leaches out of reach, it is important to plant cover crops as soon as possible after cash crops and also to use deep-rooted, fast-growing species. Cover crops must capture NO3 that is progressively moving deeper through the soil profile after cash crops stop taking up N. The deeper N is in the soil profile, the more likely it is to leach from the soil, and therefore it is particularly important for cover crops to scavenge deep soil N. Nitrogen that is captured by cover crops can be a valuable resource for farmers, if it is released into the soil as available N in synchrony with cash crop N uptake needs (Dabney, et al., 2001). However, cover crops can have detrimental effects on the environment or agronomic system if cover crop N mineralization leads to increased N leaching, or if cover crop N immobilization leads to increased fertilizer use on crops. Furthermore, unlike amending the soil with manure or compost, non-legume cover crops 138 do not add N to the soil, but rather capture N from the soil and then return the N back to the soil (Thorup-Kristensen, et al., 2003). Preemptive competition can occur, if a cover crop takes up N that would have remained in the rooting zone of the subsequent crop in the absence of the cover crop (Thorup-Kristensen, et al., 2003). While residue mineralization will affect mostly the soil surface layers, which would affect the main crop early in the growing season, preemptive competition of N resources can reduce subsoil N, which could adversely affect the main crop later in the growing season (Thorup- Kristensen, 1993). The apparent effect of cover crops will depend on the soil depth considered (e.g., examining 0-50 cm may result in very different conclusions than examining 0-150 cm). To minimize negative preemptive competition effects, the expected leaching intensity of the field and the rooting depth of the subsequent crop should be considered (Thorup-Kristensen and Nielsen, 1998). The long-term goal of using cover crops is to sustain higher levels of production with less N loss, and therefore, the efficacy of cover crops may largely depend on choosing appropriate species according to the local hydrologic regime and minimizing preemptive competition (Thorup-Kristensen, et al., 2003). For example, Thorup- Kristensen (2006a) observed that, in the spring, the subsoil (1-2.5 m) contained 120 kg N ha-1 where no cover crop had been grown but only 49 and 60 kg ha-1, respectively, where radish (Raphanus sativus L. var. oleiformis) and Italian ryegrass (Lolium multiflorum Lam.) cover crops had been grown. During the following crop season, they measured the available inorganic N in the root zone for each crop and the actual N uptake by each crop. They found that there was more available N and N uptake for leek (Allium porrum L.) after radish and leek after ryegrass in comparison to leek after no cover crop, and they 139 found there was more available N and N uptake for beet (Beta vulgaris L. var. esculenta L.) after ryegrass (they did not investigate beet after radish) in comparison to beet after no cover crop. However, the N uptake for white cabbage was decreased following ryegrass or forage radish cover crop (Thorup-Kristensen, 2006a). In the current study, we investigated the biomass N uptake, soil inorganic N depletion, and corn response following various deep-rooted cover crop systems on a broad range of soil types, geographic areas, and management regimes in Maryland and southeast Pennsylvania. Specifically, we investigated the effects of four cover crop systems—1) forage radish monoculture, 2) winter cereal monoculture, 3) winter cereal + forage radish + legume mixture, 4) no cover crop control on: 1) Inorganic N distribution within the upper 210 cm of soil in late-fall and early- spring; 2) Cover crop biomass and N uptake 3) Corn biomass and N content, and soil (0-30 cm) NO3-N and NH4-N concentration in June (corn growth stage V5); 4) Corn yield, with various N fertilizer rates. Materials and Methods Locations Cover crop experiments were conducted on 19 farm sites, two of which were at a University of Maryland dairy farm (Central Maryland Research and Education Center, Clarksville, MD) with the other 17 being on private commercial farms. Experiments were located throughout the main agricultural areas in Maryland and Southern Pennsylvania. 140 Appendix 7, Table 27 lists soil characteristics including pH, percent sand, percent clay, percent C, and percent N of the study site soils for each 30 cm depth increment from 0- 210 cm, and percent soil organic matter (SOM), and P, K, Mg, Ca, and S (mg kg-1) for 0- 30 cm. Cover crop experimental design and treatments Depending on the farmers’ preferences, situation and facilities, the cover crop experiments varied somewhat among sites, with regard to plot size, specific cereal species used, tillage practices and planting dates. In general, the experiments followed a randomized complete block design with three to four blocks. Plot size was dependent on the equipment and land available on a given farm, and were on average 409 m2, ranging from 45 m2 to 2128 m2 (Table 19). Cover crop treatments typically included 1) forage radish (radish), 2) winter cereal (cereal), 3) a multi-species cover crop comprised of forage radish + winter cereal + crimson clover (Trifolium incarnatum L.) (mix), and 4) a control of winter weeds only with no-cover crop planted (control). The winter cereal species and species in the cover crop mix varied according to farmer preference. Lancaster IB and Kent II farms also had a late-planted cover crop mix treatment, which was planted 2-4 weeks after the other cover crops. Table 18 indicates site histories of the cover crop experiment sites. Table 19 indicates cover crop treatments, planting dates, management details, and weather details. Corn response experimental design and treatments On nine of the farms with cover crop experiments, corn was planted following cover crop termination to test for cover crop effects on V5 corn growth and/or corn yield. On five farms the fall cover crop treatment main plots were split, at corn planting, into 141 multiple N fertilizer rate sub plots (Table 20), and the response of corn to N fertilization was measured. Table 20 describes the corn planting, N fertilization, herbicide, harvest, and sampling regime in each experiment. Biomass and soil sampling and analysis Cover crop biomass samples and soil cores from 0-210 cm deep were obtained in late-fall, prior to the cover crop species dying or becoming dormant for the winter, and in late-spring, shortly before cover crop termination. Biomass was collected from two to five 0.25 m2 quadrats per plot (Appendix 8, Table 28). Quadrats were randomly placed in a plot, approximately equal distance apart, one near each end and the others near the middle. No samples were taken within one meter of the plot boundaries to avoid edge effects. Winter cereal species were harvested 1 cm above the soil surface. Forage radish was harvested by hand-pulling the fleshy root from the soil. The radish leaf and root were separated. Radish roots were thoroughly washed to remove all soil. In the mixture cover crop plots, the radish, winter cereal, and legume species were separated. The legume was harvested 1 cm above the soil surface. For all treatments, weeds were separated from cover crops. Weeds were only collected if they were estimated to be > 5% of the total cover crop biomass. In the lab, radish roots were chopped into approximately 2 cm3 pieces to expedite drying. Biomass samples were dried in paper bags at 45 oC until a constant weight was attained. Dry biomass weights were recorded. The biomass was ground to 1 mm sieve size. Biomass samples were analyzed for total C and N at University of Maryland Department of Environmental Science and Technology Analytical Lab (LECO CN628 Elemental Analyzer LECO Corp., St. Joseph, MI). 142 The biomass of each tissue type (radish shoot, radish root, winter cereal shoot, legume) was converted to kilograms per hectare (Equation 15). Cover crop biomass was multiplied by the percent N to get the cover crop N content, and the amount of N taken up by cover crops was converted to kilograms per hectare (Equation 16). For radish and mixture treatments, the biomass and the N content of the tissue types were summed to determine the cover crop biomass per hectare. Weeds were not included in the biomass calculation if they were < 5% of the total biomass in a quadrat. Equation 15. Cover crop biomass per hecatare kg biomass per species g biomassQuad 1 g biomassQuad 2 10,000m 2 1 kg = ( 2 + ) ∗ ∗ ha 0.25 m 0.25 m2 1 ha 1000 g Equation 16 Cover crop N per hectare % N % N 𝑘𝑔 𝑁 𝑝𝑒𝑟 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 (g biomass∗ )100 𝑄𝑢𝑎𝑑 1 (g biomass∗ ) 100 𝑄𝑢𝑎𝑑 2 10,000 𝑚 2 1 𝑘𝑔 = ( 2 + 2 ) ∗ ∗ ℎ𝑎 0.25 𝑚 0.25 𝑚 1 ℎ𝑎 1000 𝑔 Growing degree days (GDD) and precipitation available to cover crops were determined between cover crop planting date and fall or spring cover crop sampling, based on the precipitation and temperature data from the closest weather station to the study site (Appendix 9, Table 29). Growing degree days were calculated using Equation 17. The base temperature used was 4.4°C. While the base temperature of 10°C is typically used for corn growth (http://www.cpc.ncep.noaa.gov/products/analysis_monitoring/cdus/degree_days/gdd.sht ml), lower temperatures are often used when studying cover crop growth—e.g., 4.4°C by Mirsky, et al. (2011), 5°C by Komainda, et al. (2016) and Schroder, et al. (1996), and 0°C by Farsad, et al. (2011) and Tribouillois, et al. (2016). Equation 17 Growing degree days 143 GDD = ∑P-S ((daily max temperature (°C) + daily min temperature)/2) – 4.4°C Where, P = planting date, and S = sampling date Soil cores were taken by hand-driving a Veihmeyer probe (Veihmeyer, 1929) into the ground using a 6.8 kg drop hammer. Cores were taken from 0 to 210 cm deep when possible, or until the probe reached groundwater or an impenetrable layer of rock. After the probe was driven into the ground, a jack and lever system was used to remove the probe from the ground. In the experiments conducted in the 2014-2015 season, two soil cores were taken per plot. In the fall, the two cores were taken approximately 60 cm apart, and in the spring the two cores were taken on opposite ends of the plot. Soil was extracted in two increments, from 0-120 cm deep, and from 120-210 cm deep. Soil cores were emptied into a trough and arranged, as necessary, to the appropriate length in the trough (e.g., to 120 cm for 0-120 cm extraction and to 90 cm for the 120-210 cm extraction). Soil cores were divided into 15 cm increments. For each 15 cm depth increment the two soil cores taken per plot were combined into one composite sample. In the 2015-2016 experiments, in the fall and spring, three to five 210 cm soil cores were taken per plot. Lancaster V had 5 in the fall and three in the spring; Dorchester IB had four in the fall and three in the spring; Frederick IV had five in the fall (when possible, very rocky soil); Howard IB had three in the spring; Lancaster IB had three or four in the spring; Kent II had three in the spring. The cores were approximately equally spaced throughout the plot. Soil was extracted in three increments, from 0-60 cm deep, from 60-120 cm deep, and from 120-210 cm deep. Soil cores were emptied and arranged to the appropriate length in the trough. Soil cores were divided into 30 cm 144 increments. On Dorchester IB farm, soil cores were taken only to 180 cm, and the 120- 180 soil core was kept as a 60 cm increment, rather than being divided into 30 cm increments. On Kent II farm, soil cores were taken only to 180 cm, and the 60-120 cm and 120-180 soil cores were kept as a 60 cm increment, rather than being divided into 30 cm increments. The multiple cores per plot were combined into one composite sample for each depth increment. On six farms, corn plants and soil cores were sampled in June when corn reached the V5 (five leaf collar) growth stage and at least 30 cm tall (Table 20). Soil sampling was based on the procedures for pre-sidedress nitrate testing (PSNT) (Cornell University Cooperative Extension, 2012). Soil cores were taken at eight points per plot (or subplot when applicable). Areas within the 1 m edge of the plot were avoided. Soil cores were placed randomly (not all in corn rows or between corn rows). Soil cores were taken from 0-30 cm deep. The soil cores were mixed and a composite sample was collected. The closest corn plant to every soil sample was collected. Corn plant height measurements were taken from the soil to leaf height. When measuring the corn plant, the leaves were not pulled up; rather the height was the maximum height of the undisturbed plant. At the Kent II site, sections of corn rows were sampled rather than randomly located plants. Four meters of corn plants per plot were sampled (approximately 28 plants). A 1 m segment from the two center rows of corn in a plot, on both ends of the plot were sampled. The 1 m segments were 1 m in from edge of plot. Corn plant height was not recorded. 145 Corn plants were cut 1 cm from the soil. The corn was dried at 40 °C until it reached a constant weight. The corn was ground to 1 mm sieve size. For the Kent II farm, only 1/3 of the corn samples (about 9 plants) were ground. Biomass samples were dried in paper bags at 40 oC until a constant weight was attained. Dry biomass weights were recorded. The biomass was ground to 1 mm sieve size. Biomass samples were analyzed for total C and N at University of Maryland Department of Environmental Science and Technology Analytical Lab (LECO CN628 Elemental Analyzer LECO Corp., St. Joseph, MI). In the field, the soil samples were put into sealed plastic bags and stored on ice for transport to the lab. The soil samples were dried at 40 °C for at least 48 hours, and the soil was sieved through a 2 mm sieve. The weight of the soil at the field moisture level, the weight of the soil after drying, and the weight of the gravel that did not pass through the 2 mm sieve was determined. Exchangeable NO3 and NH4 in the soil was extracted with 0.5 M potassium sulfate (K2SO4) solution. Two grams of dry soil were mixed with 20.0 ml of 0.5 M K2SO4 in 50 ml tubes. The tubes were shaken at 200 rpm for 30 minutes and then settled undisturbed for 10 minutes. The liquid from the tubes was filtered through VWR 410 filter paper. The filtrate was tested for NO3-N and NH4-N using a Lachat QuikChem 8500 Automated Ion Analyzer (Hach Company, Loveland, CO). The filtrate was analyzed for NH4–N by the salicylate method and for NO2-N and NO3–N by cadmium reduction method. The measured NO3-N and NH4-N (mg NO3-N L -1 or mg NH -14-N L ) was blank-corrected with filtered 0.5 M K2SO4 solution samples and converted to mg NO3-N or NH4-N kg soil -1 (Appendix 3). 146 In order to convert values of NO3-N and NH4-N concentrations in the soil to stock amounts of NO3-N and NH -1 4-N in kg ha , soil bulk density values were estimated from dry mass of known soil volumes in the cores and corrected for gravel content (Equation 18). The mass and volume of soil was determined for each of the soil cores taken with the Veihmeyer probe. Bulk density values for each farm were based on the average of all cores from that farm for a given depth increment (e.g., 0-120 cm or 120-210 cm) (Appendix 10). Equation 18 Bulk density of soil 𝑔 𝑠𝑜𝑖𝑙 (𝑔 𝑠𝑜𝑖𝑙+ 𝑔𝑟𝑎𝑣𝑒𝑙)−𝑔 𝑔𝑟𝑎𝑣𝑒𝑙 3 = 𝑐𝑚 𝑔 𝑔𝑟𝑎𝑣𝑒𝑙 (𝜋𝑟2∗ℎ𝑒𝑖𝑔ℎ𝑡)−( ) 2.65 𝑔 𝑐𝑚−3 Where, r = radius (in cm) of soil core, as determined by measuring the inside diameter of soil core tip to three significant figures and dividing by 2. height = length (in cm) of the increment of soil collected estimated bulk density of gravel = 2.65 g cm-3 Farm topsoil (0-30 cm) amounts of NO3-N and NH4-N (kg N ha -1) and sand/clay/silt fractions was determined on late-summer samples, prior to establishing the cover crop treatments, with the exceptions of four farms: for Lancaster II and Huntington IA November control plot samples were analyzed, for Frederick III and Lancaster III April control plot samples were analyzed. The pH was analyzed by a glass combination pH electrode and a pH meter (Metler Toledo InLab®413 combination meter). Soil particle size analysis was performed according to the modified pipette method (Gavlak, et al., 2005). Total C and N analysis 147 was performed at University of Maryland Department of Environmental Science and Technology Analytical Lab on LECO CN628 Elemental Analyzer (LECO Corp., St. Joseph, MI; Nelson and Sommers, 1996; Matejovic, 1993). Soil organic matter (SOM) (Loss on Ignition Method) and nutrient content by Mehlich3 extraction (P, K, Mg, Ca, Na, S) was measured at WayPoint Analytical, Inc (Richmond, VA). Corn silage and grain yield Corn grain or silage yield was estimated by taking hand-samples or using a harvester (Table 20). When corn silage or grain was estimated by taking hand-samples, the harvest area per plot was two rows by 6.10 m long. The harvest area was at least 1.52 m from plot ends and there were at least two border rows of corn between the edge of the plot and harvested row. For corn silage harvest, corn plants were cut 4 cm from the ground and weighed using a hanging scale and tarp. From the harvested corn plants, a subsample of 6-11 plants was selected. For each subsample, the stover (including stalk and ear husk) and corn ears (grain + cob) were weighed separately, then dried at 40 °C and re-weighed. The wet and dry weights for each subsample were used to determine the whole plant percent moisture. The corn silage yield was estimated by dividing the whole plant dry weight by the harvest area (Appendix 11, Equation 19). For the corn grain yield measurements, within the harvest area the corn ears were husked and broken off from the plant (leaving the husk attached to the stover). The ears were weighed. From the harvested ears, seven to nine ears were randomly selected as a subsample and weighed. The ears were brought back to the lab and dried at 40 °C until the weights remained constant. The dry weights were determined for corn ears. The wet 148 and dry weights for the corn ears were used to determine the ear percent moisture. The proportion of grain in the ear and the percent moisture of the ear were used to estimate the dry grain weight adjusted to 15.5% moisture. The corn grain yield was estimated by dividing the grain weight at 15.5% moisture by the harvest area (Appendix 11, Equation 20). Statistical Analyses All analyses were performed using SAS version 9.4 statistical software (SAS Institute, Cary, NC). The level of probability considered significant was p < 0.05, unless otherwise stated. Using Proc Mixed, an analysis of variance (ANOVA) was performed for fall and spring measurements of cover crop parameters (biomass, N content, C/N ratio) and soil parameters (amounts of NO3-N and NH4-N for 0-90 cm soil, 90-210 cm soil, and every 30 cm soil layer increment from 0 to 210 cm deep). Cover crop treatment was the fixed effect. Block or block within site was the random effect. Analyses were performed 1) separately for each site, 2) across sites for fall measurements (six sites for soil parameters, 13 sites for cover crop parameters), and 3) across sites for spring measurements (11 sites for soil parameters, 11 sites for cover crop parameters). Using Proc Corr, a Pearson product-moment correlation was performed relating cover crop biomass (fall radish, fall winter cereal, fall + spring winter cereal) to weather factors (GDD, precipitation) and soil characteristics (topsoil NO3-N and NH4-N, topsoil percent sand, clay, and silt). For each corn trial experiment site in which V5 corn and PSNT soil samples were taken, an ANOVA was performed investigating if the independent variable of cover crop treatment affected V5 corn biomass, V5 corn N content, and 0-30 cm soil NO3-N and 149 NH4-N concentrations. Across the six sites that had V5 corn and PSNT soil samples taken, these same variables were analyzed with rep(site) as a random factor in the analysis. For each site at which corn yield samples were taken, an ANOVA was performed investigating if the independent variable of cover crop treatment, and in some cases the independent variable of fertilizer N rate and the interaction of cover crop treatment x fertilizer N rate, affected corn yield. Across the six sites that had corn grain samples taken in plots with the normal N fertilizer rate of the farm, and the six farms that had corn grain samples taken in the no fertilizer plots, an ANOVA was performed, with site(rep) as a random factor in the analysis, investigating if the independent variable of cover crop treatment affected the dependent variable of corn yield. Results Cover crop effects on soil inorganic N 0-210 cm deep In late-fall, across six farms, for the 0-30 cm and 30-60 cm deep soil increments, the NO -N (kg ha-13 ) was significantly higher in the control treatment than the radish, winter cereal, or mix treatments, and for the 60-90 cm soil increment, NO -13-N (kg ha ) was significantly higher in the control treatment than the radish treatment. In late-fall, for the 0-30 cm deep soil increment, the NH -N (kg ha-14 ) averaged across six farms was significantly lower in the control treatment than the mix treatment (Table 21). In the spring, across 11 farms, in every 30 cm soil increment from 0-210 cm, soil in the winter cereal treatment had significantly lower NO3 than soil in the control treatment. In soil increments from 30-210 cm deep, soil in the mix treatment had 150 significantly lower NO3 than soil in the control treatment, and the same level of NO3 as soil in the winter cereal treatment; from 0-30 cm deep, soil in the mix treatment had the same level of NO3 as soil in the control and radish treatments and higher NO3 than soil in the winter cereal. From 0-30 cm deep, soil in the radish treatment had significantly higher NO3 than soil in control or winter cereal. From 30-60 cm deep, soil in the radish treatment had significantly higher NO3 than soil in winter cereal or mix treatments (and the same level as control). In each 30 cm increment from 60-150 cm deep, soil in the radish treatment had significantly higher NO3 than soil in the winter cereal, the same level of NO3 as soil in mix, and lower NO3 than soil in control. In each 30 cm increment from 150-210 cm deep, soil in radish had significantly higher NO3 than soil in control and the same level of NO3 as soil in mix and winter cereal. Across 11 farms, the soil NH4-N (kg ha -1) did not differ at any soil depth increment (Table 21). Site by site findings The fall and spring soil NO3-N and NH4-N from 0-90 cm and 0-210 cm for each farm site is listed in Table 22. Figure 15 shows cover crop N uptake and the November residual soil NO3-N levels for late-fall and/or spring samples. Soil nitrate by depth increment Dorchester IB—In the fall, there were no differences among cover crop treatments for soil NO3-N in any 30 cm layer. In the spring, control had more NO3-N than mix in soil layers from 30-120 cm, control had more NO3-N than winter cereal in soil layers from 30-90 cm, and control had more NO3-N than radish in soil layers from 60- 120 cm (Figure 16). Frederick I—In the fall, control had more NO3-N than mix in soil layers from 60-120 cm. (No fall soil samples were taken in radish or winter cereal.) In the 151 spring, control had more NO3-N than radish, winter cereal, and mix in 30-60 cm soil, and control had more NO3-N than winter cereal and mix in the 60-90 cm soil (Figure 16). Frederick III—In the spring, control had more NO3-N than radish, mix, or winter cereal in soil layers from 30-60 cm deep (Figure 16). Frederick IV—In the fall, control had more NO3-N than radish or winter cereal in soil layers from 0-90 cm (Figure 16). Harford I—In the fall, control had more NO3-N than radish or mix in soil layers from 0- 60 cm, and radish had more NO3-N than mix and control in 180-210 cm soil (Figure 16). Howard IA— In the fall, there were no differences among cover crop treatments for soil NO3-N in any 30 cm layer (although only two replications were sampled). No soil samples were taken in the spring. Howard IB—In the spring, radish had more NO3-N than mix, control, and winter cereal, and control and mix had more NO3-N than the winter cereal in the 0-30 cm soil. Radish had more NO3-N than mix, control, and winter cereal in the 30-60 cm soil. Control and radish had more NO3-N than winter cereal, and control had more NO3-N than mix in 60-90 cm soil. Control had more NO3-N than winter cereal in 90-120 cm soil (Figure 16). Huntington IA—In the fall, control had more NO3- N than radish, winter cereal, and mix in 0-30 cm soil. In the spring, mix had more NO3-N than control, and mix, radish, and control had more NO3-N than winter cereal in 0-30 cm soil. Mix and control had more NO3-N than winter cereal in 30-60 cm soil. Control had more NO3-N than winter cereal in soil layers from 0-90 cm and 120-180 cm (Figure 16). Kent II— In the spring, there were no differences among cover crop treatments for soil NO3-N in any 30 cm layer. No soil samples were taken in the fall. Lancaster IA—In the fall, control had more NO3-N than winter cereal in soil layers from 30-90 cm deep. Control had more NO3-N than radish and mix in 30-60 cm soil. In the spring, control had 152 more NO3-N than winter cereal, radish, and mix in the soil layers from 60-150 cm and 180-210 soil (Figure 16). Lancaster IB—In the spring, radish and control had more NO3-N than winter cereal, mix and late-mix in 30-60 cm soil. Control had more NO3-N than radish, winter cereal, mix and late-mix in soil layers from 60-120 cm (Figure 16). Lancaster II—In the fall, control had more NO3-N than winter cereal and radish in soil layers from 0-60 cm, and control had more NO3-N than mix in 0-30 cm soil. In the spring, control had more NO3-N than winter cereal in soil layers from 30-120 cm, control had more NO3-N than mix in soil layers from 30-90 cm deep, and control had more NO3- N than radish in 60-90 cm soil (Figure 16). Lancaster III—In the spring, radish had more NO3-N than mix in soil layers from 0-60 cm. radish and control had more NO3-N than winter cereal and mix in soil layers from 90-150 cm. Radish had more NO3-N than mix in 150-180 cm soil (Figure 16). Lancaster V—In the fall, control had more NO3-N than winter cereal, mix, and radish in soil layers from 0-120 cm deep. In the spring, radish had more NO3-N than winter cereal and control in soil layers from 0-60 cm. Control had more NO3-N than radish and winter cereal from 60-90 cm. control had more NO3-N than winter cereal in soil layers from 90-120 cm. Radish had more NO3-N than winter cereal in 120-150 cm soil. Radish and control had more NO3-N than winter cereal in 150-180 cm soil (Figure 16). (No spring soil samples were taken in mix.) Soil ammonium by depth increment Lancaster IA, Lancaster II, and Lancaster V were the only farms out of the 14 to have significant differences in NH4-N at any depth increment. For Lancaster IA, in the fall, there were no differences between cover crop treatments for NH4-N levels. In the spring 0-180 cm soil, the control treatment had higher NH4-N than the triticale treatment. 153 For Lancaster II, in the fall, in the soil layers from 0-120 cm, the mixed species cover crop treatment had significantly more NH4-N than the control and radish treatments, and from 30-60 cm and 90-120 cm more than the triticale treatment. From 180-210 cm deep, the triticale treatment had significantly more NH4-N than the radish and control treatments. In the spring, there were no differences between cover crop treatments for NH4-N levels. For Lancaster V, in the fall, in soil layers 0-30 cm and 90-120 cm, the mixed species cover crop treatment had significantly more NH4 than the control and radish treatments. In the spring, there were no differences between cover crop treatments for NH4 levels. Dorchester IB, Frederick I, and Huntington IA, farms had no differences between cover crop treatments at any soil depth for NH4-N amounts in the fall or spring. Frederick IV, Harford I, and Howard IA farms had no differences between cover crop treatments at any soil depth for NH4-N amounts in the fall and samples were not taken in the spring. Frederick III, Howard IB, Kent II, Lancaster IB, and Lancaster III farms had no differences between cover crop treatments at any soil depth for NH4-N amounts in the spring and samples were not taken in the fall. Cover crop growth and N content The fall and spring cover crop biomass and cover crop N content for each farm site is indicated in Table 22. In the fall, across the 13 farms, radish biomass was significantly higher than mix, which was significantly higher than winter cereal. The N content was significantly higher for radish and mix than for winter cereal. The C/N ratio did not differ between radish, mix and winter cereal cover crop treatments (Table 23). 154 In the spring, across the 10 farms, winter cereal biomass was higher than mix. There were no significant differences between the N uptake of winter cereal and mix. The C/N ratio was greater for winter cereal than mix (Table 23). When comparing radish to winter cereal prior to their termination (i.e., naturally winter-killing for radish or oat (Avena sativa L.) and chemically terminated with herbicide for rye (Secale cereale L.) and triticale (x Triticosecale Wittm. ex A. Camus) winter cereals, biomass was not significantly different between radish and winter cereal (p = 0.9251). The N content was significantly higher for radish (80.1 kg ha-1) than winter cereal (58.7 kg ha-1) at a significance level of p = 0.0013. The C/N ratio was significantly lower for radish (14.2) than winter cereal (22.6) at a significance level of p < 0.0001 (Table 23). Relationships between cover crop biomass and environmental characteristics The number of GDD was positively correlated to fall radish biomass (r = 0.43; p = 0.094) and spring winter cereal biomass (r = 0.63, p = 0.012). Precipitation was positively correlated to spring winter cereal biomass (r = 0.58; p = 0.025). Topsoil (0-30 cm) NO3-N was positively correlated to fall winter cereal biomass (r = 0.77; p = 0.001) and to fall radish biomass (r = 0.48; p = 0.080). The percent sand in the topsoil was negatively correlated to fall winter cereal biomass (r = -0.52; p = 0.056), fall radish biomass (r = -0.47; p = 0.092), and spring winter cereal biomass (r = -0.74; p = 0.002). Topsoil nitrate was negatively correlated with topsoil percent sand in fall radish plots (r = -0.50; p = 0.070), fall winter cereal plots (r = -0.53; p = 0.050), and spring winter cereal plots (r = -0.57; p = 0.026). The topsoil percent silt was positively correlated to fall 155 winter cereal biomass (r = 0.52; p = 0.0582), fall radish biomass (r = 0.56; p = 0.0381), and spring winter cereal biomass (r = 0.70; p = 0.004) (Table 24). Cover crop effect on PSNT soil and corn plants Across the six farms on which PSNT soil samples (30 cm deep at corn V5 stage) were taken, the soil NO3-N concentration for radish was significantly higher than for winter cereal and mix. The soil NH4-N concentrations did not differ among any of the cover crop treatments. At the Howard IB site, radish NO3-N concentration was higher than winter cereal, and at the Kent II site radish NO3-N concentration was higher than mix and late-mix. Across the six farms, the V5 corn biomass plant-1 and shoot N plant-1 were significantly affected by the previous cover crop treatment in the order radish > mix = control > winter cereal (Table 25; Figure 18). Cover crop effect on corn yield and corn response to N fertilizer Averaged across six farms at the farmers’ standard N fertilizer application rate, corn yield following radish or control was higher than winter cereal. Corn yield following radish was higher than mix. Averaged across six farms at the 0 fertilizer N rate, corn yield following radish > control = mix > winter cereal (Table 26). For Franklin IIB (0 fertilizer N rate) and for Frederick IV (farmer’s standard fertilizer N rate), there was no significant cover crop treatment effect on corn silage yield. For Howard IA, there was a corn grain yield response to cover crop treatment and N fertilizer treatment, but no interaction. Corn in radish and control yielded more than corn in winter cereal. Corn yield responded to fertilizer N in order of 0 kg ha-1 < 67 kg ha-1 < 157 kg ha-1 = 202 kg ha-1 (Figure 19). For Howard IB, there was a corn grain yield response to cover crop treatment, N fertilizer treatment, and the interaction between cover 156 crop x N fertilizer (p = 0.0889). At all fertilizer rates, corn in radish yielded higher than corn in winter cereal, and at some fertilizer rates corn in radish yielded higher than corn in mix or control. At 67 kg ha-1, 135 kg ha-1, and 202 kg ha-1 fertilizer rates, corn in mix yielded higher than corn in winter cereal. Corn yields following radish and control did not increase between N application levels of 135 and 202 kg ha-1 fertilizer N. Corn yields following winter cereal and mix (p = 0.055) increased with 202 kg ha-1 fertilizer N in comparison to 135 kg ha-1 fertilizer N (Figure 19). For Frederick I, there was a corn grain yield response to cover crop treatment and N fertilizer treatment, but no interaction. Corn in radish and control yielded more than corn in winter cereal, and corn in radish yielded more than corn in mix. Corn yield responded to fertilizer N in order of 0 kg ha-1 < 112 kg ha-1 = 168 kg ha-1 (Figure 19). For Lancaster IA, there was a corn grain yield response to the interaction between cover crop x N fertilizer. In the 0 kg ha-1 fertilizer N treatment, corn in winter cereal yielded less than corn in radish, mix, or control. Also, corn yield responded to fertilizer N in order of 0 kg ha-1 < 140 kg ha-1 = 168 kg ha-1 = 224 kg ha-1 in winter cereal. Corn in radish, mix, and control did not respond to fertilizer N (Figure 19). For Lancaster IB, there was a corn grain yield response to cover crop treatment and N fertilizer treatment, but no interaction. Corn in radish and control yielded more than corn in winter cereal, and corn in radish yielded more than corn in early-planted mix. Corn with 135 kg N ha-1 yielded more than corn with 67 kg N ha-1 (Figure 19). Discussion Cover crop effects on soil inorganic nitrogen 0-210 cm deep 157 Radish, winter cereal, and mix cover crops each performed differently in terms of reducing deep soil NO3 in the fall and spring and increasing surface soil NO3 in the spring. Radish was the most effective cover crop at reducing the soil NO3 in the fall from the deeper soil layers and ensuring available NO3 on the soil surface (0-30 cm) in the spring. In the fall, radish reduced NO3 pools to 90 cm deep, while winter cereal or mix cover crops reduced NO3 to only 60 cm deep. Other studies have also found radish to be more effective than rye at reducing levels of soil NO3-N by late-fall, especially in deep layers (> 1 m) (Thorup-Kristensen, 2001; Thorup-Kristensen, et al., 2009; Kristensen and Thorup-Kristensen, 2004a). However, in the spring, radish was less effective than winter cereal at reducing soil NO3 from 30-150 cm deep. Winter cereal was the most effective at reducing soil NO3 in the spring throughout the entire soil profile. Mix was more effective than winter cereal and as effective as radish at ensuring available NO3 on the soil surface (0-30 cm) in the spring, and was as effective as winter cereal in reducing soil NO3 from 30-210 cm soil. Several other studies found that in the spring, rye or radish cover crops did not reduce upper layer (0-0.5 or 1 m) soil NO3-N content compared to the no cover crop control, but they did reduce deep soil (> 1 m) soil NO3-N content (Thorup-Kristensen, 2006b; Thorup-Kristensen, et al., 2009). At most sites in the current study, soil NH4-N did not differ between cover crop treatments. Other studies have found that NH4-N levels in the soil tend to be less variable among cover crop or crop species than NO3 levels (Kristensen and Thorup-Kristensen, 2004a; Kristensen and Thorup-Kristensen, 2004b). For example, Bergstrom (1986) took soil cores 1 m deep six to nine times per year in cropping systems of barley (Hordeum 158 vulgare L.), barley with 120 kg N ha-1 fertilizer, fescue (Festuca L.) with 200 kg N ha-1 fertilizer, and alfalfa (Medicago sativa L.) with no fertilizer. Ammonium amounts did not vary much between treatments, staying between 11 and 13 kg N ha-1, whereas NO3-N ranged between 23 and 68 kg N ha-1 (Bergstrom, 1986). On some sites in the current study, NH4-N was higher in cover crop treatments than control no cover crop. Lacey and Armstrong (2015) found that the NH4-N of radish and rye cover crops was higher than no cover crop in the spring of one of two study years, while the other year there were no differences, possibly attributable to cold and wet soil conditions slowing mineralization rates. Cover crop growth and N content In terms of fall growth, radish was more productive than winter cereal or mix cover crops. Kristensen and Thorup-Kristensen (2004a) and Thorup-Kristensen (2001) also found radish had higher biomass and N accumulation than rye. We found that the radish biomass was weakly correlated with GDD, while fall winter cereal biomass was not correlated to GDD. Radish is more sensitive to cold weather than winter cereal cover crops (Lacey and Armstrong, 2015; Thorup-Kristensen, et al., 2003). As expected, spring winter cereal biomass was correlated to GDD. Both radish and winter cereal biomass increased with lower percent sand and more NO3 in the topsoil, which was expected as NO3 would more readily leach from sandy soils and become less available to support cover crop growth. Radish winter-killed at all farm sites. For samples taken shortly before winter-kill, the average C/N ratio was 14.2. When C/N ratios are < 25/1, N will generally be plant available and not immobilized (Weil and Brady, 2017). Therefore, N released from the 159 dead radish biomass was likely available for plant uptake and not immobilized. The cover crop biomass shortly before termination for radish and winter cereal did not differ, but on average radish had accumulated 21.4 kg ha-1 more biomass N than winter cereal, and radish biomass had a C/N ratio of 14.2 while the winter cereal C/N ratio was 22.6. Therefore, the radish cover crop had both greater N accumulation in the biomass and a lower likelihood of N immobilization due to the lower C/N ratio. Winter cereal cover crops commonly have C/N ratios higher than mixes. On a loamy sand soil in North Carolina, the C/N ratio for October-planted cover crops in April was greatest to smallest in the order of rye monoculture (38) > rye/clover mix (27) > crimson clover monoculture (16) (Ranells and Wagger, 1997). For a hairy vetch/rye mix cover crop, as vetch went from comprising 0 to 100% of the mixture, the N content increased (64 to 181 kg N ha-1) and C/N ratio decreased (83 to 16) (Poffenbarger, et al., 2015b). Corn yield had a negative linear relationship with cover crop C/N ratio (R2 = 0.55) and a positive relationship with cover crop biomass (R2 = 0.23) (Finney, et al., 2016). White, et al. (2016) found that to maximize cover crop N supply and provide the greatest yield benefit to a subsequent corn crop, cover crops should have low C/N ratio and high biomass N content. Species that fit these criteria, based on experiments performed in Pennsylvania, include legumes such as fava bean (Vicia faba L.), red clover (Trifolium pretense L.), and hairy vetch (Vicia villosa Roth.), grown in monoculture or in mixtures with each other or with grasses including triticale, Italian ryegrass, or oat or a brassica forage radish. Thomsen, et al. (2016) found during incubation studies with forage radish, white mustard (Sinapis alba L.), and perennial ryegrass using a loamy sand soil, that the residue C/N ratio and N concentration were the best single predictors for net 160 N mineralization, regardless of temperature, or of cover crop type, age, or planting date. It is important to note that the timing of the mineralization is also important, since it is possible that N could mineralize and leach out of the rooting zone before spring crops take it up. Corn growth and yield In June, at PSNT sampling, June soil NO3-N concentrations were positively related to the fraction of total corn yield. Corn with a preceding rye cover crop tended to have lower soil NO3-N concentrations and corn yields, while corn with a preceding radish cover crop tended to have higher soil NO3-N and corn yields (Figure 17). The critical value for PSNT NO3-N concentrations is 21 mg N kg -1 soil. If the NO3-N concentration is greater than 21 mg N kg-1 soil, sidedress N fertilizer is not recommended in Maryland (University of Maryland Extension, 2010). The corn following all of the cover crop treatments fell below this threshold and required N sidedress application, but the radish treatment had higher topsoil NO3-N than either the cereal or cover crop mixtures. Nitrogen fertilizer response curves can help determine if more or less fertilizer is required for optimal corn yields. At Howard IB farm, the N response plateaued for corn following radish cover crop, mix cover crop, or control cover crop at 135 kg ha-1 N applications (farmer’s normal rate), but the N response continued to increase for corn following rye cover crop even at 202 kg ha-1 (150% of farmer’s normal rate). The Lancaster IA farm had outstanding soil fertility and corn was minimally responsive to N fertilization in any of the treatments. The Howard IA corn showed minimal responses to N as well. On Frederick I farm, there was not a significant interaction between corn yield and fertilizer rate, although corn following radish and control had yields around 10 Mg 161 ha-1 that leveled off at 112 kg ha-1 N fertilizer application (100% farmer’s normal rate), while corn following winter cereal had maximum yields of 7.6 Mg ha-1 at 168 kg ha-1 N fertilizer application (150% of farmer’s normal rate). While corn N fertilizer response is variable based on site fertility, in our study, corn following winter cereal tended to have lower yield and/or higher fertilizer requirements than corn following no cover crop or a radish or mixed cover crop. A meta- analysis including 47 studies with grass cover crops and 13 studies with mixed species cover crops found that corn following a mixed cover crop had 13% higher average yields than no cover crop, and mixed cover crops with late termination dates (0-6 days before corn planting) had 30% higher corn yield compared to no cover crop (Marcillo and Miguez, 2017). Corn following a grass cover crop was not different than no cover crop, however management practices such as the corn N fertilizer rate was a highly significant moderator of yield response (Marcillo and Miguez, 2017). Nitrogen in winter cereal cover crops is released very slowly by decomposition and is often immobilized by microbes utilizing the abundant carbon in the residues and is therefore largely unavailable for crop uptake (Adeli, et al., 2011; Doran and Smith, 1991; Ketterings, et al., 2015; Thorup‐Kristensen and Dresbøll, 2010). As a result, higher levels of spring N fertilizer are often applied following winter cereal cover crops than would be applied without a cover crop. We found that in some cases, radish improved corn yield in comparison to the no cover crop control. On a Hadley fine sandy loam soil in Massachusetts, Jahanzad, et al. (2017) found that potato (Solanum tuberosum L.) yield and yield components were higher for potato following forage radish cover crop than cereal rye or no cover crop. 162 Potato grown following forage radish produced the highest yield when fertilized with 75 or 150 kg N ha -1, while potato grown following no cover crop produced the highest yield when fertilized with 225 kg N ha -1 (Jahanzad, et al., 2017). In contrast, studies performed in Minnesota, Wisconsin, and Missouri indicated no fertilizer replacement value or benefit on corn yield of forage radish, despite substantial N uptake by the radish (Gieske, et al., 2016; Sandler, et al., 2015; Ruark, et al., 2018). Conclusions and practical applications Through performing on-farm trials of early-planted cover crop systems, we were able to observe a range of cover crop responses. Cover crops are affected by soil type, management, and weather. Furthermore, site-by-site results of residual soil NO3-N and NH4-N were often highly variable. Soil sampling can be challenging due to the heterogeneity of soil, more so in deep soil layers. Even with the variability among sites and soil samples, there were clear trends showing that early-planted forage radish and rye cover crops (monoculture or mix) can scavenge soil N from 1+ m. This is expected to reduce NO3-N leaching. Overall it can be concluded that winter cereal had a negative impact on the following corn. Either extra fertilizer will need to be added, which is contrary to the goal of improving the overall cropping system’s nutrient use efficiency, or farmers’ yields will be reduced, which is contrary to the goal of “making cover crops pay”. On the other hand, a mixed species cover crop has no negative (or positive) impact on the cropping system, and a radish cover crop has a neutral or sometimes positive impact on the cropping system, in terms of improving the overall nutrient use efficiency. Cover cropping is a practice that is already widely adopted in Maryland, and planting cover 163 crops earlier in the fall can greatly increase their ability to scavenge and potentially release deep soil N. 164 Table 18. Site histories of cover crop studies. Most Site Year recent Crop rotation history Manure history Tillage history crop Dorchester IB 2015-2016 wheat NA1 NA NA Franklin I 2014-2015 corn Corn, small gran silage, alfalfa Regular No-till Mostly no-till; Occasional Franklin IIA 2014-2015 corn Corn, small grain silage Regular tillage Mostly no-till; Occasional Franklin IIB 2015-2016 corn Corn, small grain silage Regular tillage Frederick I 2014-2015 corn NA Regular NA Frederick III 2014-2015 NA NA Regular NA Regular manure applications Subsoiled in 2014, disked 1x Frederick IV 2015-2016 corn Double crop corn/triticale for > 5 years spring and fall for past 10 years per year until 2016 Harford I 2014-2015 corn NA Regular NA 2014 corn silage, 2013 corn, 2012 forage Howard IA 2014-2015 corn sorghum, 2011 sweet corn, 2010 sweet Occasional No-till corn 2015 corn silage, 2014 soybean, 2013 corn No manure applications past 20+ Howard IB 2015-2016 corn grain (rye cover crop), 2012 corn grain, No-till years 2011 corn grain, 2010 corn grain Continuous corn with rye cover crop in Huntington IA 2014-2015 corn Regular No-till winter Continuous corn with rye cover crop in Huntington IB 2015-2016 corn Regular No-till winter Kent II 2015-2016 corn NA NA NA 2013 pumpkin, 2012 corn, 2011 corn, 2010 Lancaster IA 2014-2015 wheat Occasional No-till past 5+ years soybean 2014 pumpkin, 2013 corn, 2012 soybean, Lancaster IB 2015-2016 wheat Occasional No-till past 5+ years 2011 corn Tobacco plowed and Silage corn, forage rye cover crop, Regular (every year except when Lancaster II 2014-2015 tobacco cultivated several times; corn tobacco, possibly alfalfa growing tobacco) maybe no-till 165 Regular (every year except a half Silage corn, mostly grass cover crop, grain Lancaster III 2014-2015 corn rate on top of alfalfa applied No-till corn, alfalfa when dormant in fall or winter) Corn silage, forage rye, tobacco, alfalfa Mostly no-till corn, some no- Lancaster V 2015-2016 tobacco Regular rotation till tobacco 2013 soybean, 2012 corn, 2009-2011 Prince Georges I 2014-2015 corn No manure ever applied No-till past 5+ years mixed grass hay with < 25% legumes 1Data not available indicated as NA 166 Table 19. Cover crop treatments, planting date, management details, and sampling timing of cover crop studies. Plot Cover crop Cover crop Date Site size treatments with Cover crop samples Soil samples application planted seeding rates, kg ha-1 Fall Spring Fall Spring Prec., Prec., Date GDD Date GDD Date Date mm mm 69 4 Dec m x (rep 1); radish; wheat; mix 17 6.9 5 Dec 2334; (radish + annual 23 Aug Apr 28 May Dorchester IB m drilled (rep 2); 2341; 287 3560 709 4 Dec 2015 ryegrass + Crimson 2015 2016 2016 6 Dec 2349 clover); control 2015 (rep 3) 121 triticale @ 90; radish m x @ 6.7; mix (triticale drilled 15 Sep 21 Nov 1 Apr Franklin I 9.1 @ 72 + radish @ 6 + 978 153 1257 414 none none 2014 2014 2015 m crimson clover @ 12); control 15 oats @ 112; radish @ m x drilled 10 Sep 21 Nov winter Franklin IIA 6.7; mix (oats @ 90 + 1179 114 N/A N/A none none 4.6 2014 2014 kill radish @ 5.6); control m 61 oats @ 112; radish @ m x drilled 30 Aug 14 Nov winter Franklin IIB 6.7; mix (oats @ 90 + 1706 145 N/A N/A none none 4.6 2015 2015 kill radish @ 5.6); control m 49 radish @ 8.8; triticale m x @ 135; mix (triticale 5 Sep 1 Dec 9 Apr Frederick I 4.6 @ 67 + radish @ 4 + drilled 1295 193 1669 522 12/1/2014 4/13/2015 2014 2014 2015 m crimson clover @ 10); control 55 radish @ 21.4; 15 Sep 9 Apr Frederick III drilled none none none 1387 515 none 4/23/2015 m x triticale @ 135; mix 2014 2015 167 4.6 (triticale @ 67 + m radish @ 7.7 + crimson clover @ 19); control 61 7 Nov m x (rep 1- 24 7 Nov (rep 4.6 radish @ 5.6; triticale 4 Sept 3); 14 1317; 249; Frederick IV drilled Apr 2527 718 1-3); 14 Nov none m @ 146; control 2015 Nov 1389 274 2016 2015 (rep 4) 2015 (rep 4) 91 radish; mix (radish + broadcast, m x annual ryegrass + 4 Sep 16 Nov Harford I lightly 1310 168 none N/A N/A 16 Nov 2014 none 9.1 crimson clover); 2014 2014 covered m control 61 16 m x rye @ 129; radish @ 15 Sep 9 Nov Howard IA drilled 972 142 Apr 1566 632 9 Nov 2014 none 3.0 8.7; control 2014 2014 2015 m 61 radish @ 7.5; rye @ m x 139; mix (triticale @ 24 1 Sep 6 Jan 24 May Howard IB 3.0 40, radish @ 3.4, drilled 2034 410 Apr 2841 777 none 2015 2016 2016 m clover @ 6.7); 2016 control 61 radish @ 9.0; rye @ 25 m x 10 Sep 15 Nov 25 Apr Huntington IA 126; mix (oat @ 72 + drilled 935 134 Apr 1338 542 15 Nov 2014 9.1 2014 2014 2015 radish @ 5.6); control 2015 m 61 radish @ 11; ryegrass m x @ 20; mix (ryegrass 9.1 @ 5.07 + radish @ m 1.30 + crimson clover 10 Sep 6 Dec Huntington IB drilled 1223 222 none N/A N/A none none @ 12.6 + red clover 2015 2015 @ 1.95 + sweet clover @ 0.39); control 168 152 11 Sep radish; rye; mix m x 2015; 17 (radish + rye + 5 Dec 25 May Kent II 14 drilled late-mix 1588 276 Apr 2600 569 none crimson clover); 2015 2016 m 24 Sep 2016 control 2015 15 radish @ 3.4; m x triticale @ 45; 28 18 Aug 19 Dec 7 May Lancaster IA 3.0 mix (ryegrass @ 10 + drilled 2074 304 Apr 2593 583 19 Dec 2014 2014 2014 2015 m radish @ 2.2 + clover 2015 @ 4.5); control 15 radish @ 3.4; triticale 4 Sep m x @ 45; mix (triticale 2015; 24 28 Nov 24 May Lancaster IB 3.0 @ 40 + radish @ 3.4 drilled late-mix 1604 284 May 3075 820 none 2015 2016 m + clover @ 6.7); 30 Sep 2016 control 2015 31 radish @ 9.0; m x triticale @ 135; mix 11 4.6 (triticale @ 67 + 4 Sep 12 Nov 13 Apr Lancaster II drilled 1282 176 Apr 1694 574 12 Nov 2014 m ryegrass @ 22 + 2014 2014 2015 2015 radish @ 5.6 + clover @ 17); control 31 radish @ 9.0; triticale m x @ 135; mix (triticale 27 11 Sep 27 Apr Lancaster III 4.6 @ 67 + ryegrass @ drilled none N/A N/A Apr 1617 630 none 2014 2015 m 22 + radish @ 5.6 + 2015 clover @ 17); control 15 6 Nov m x radish @ 9.0; triticale 2015 6 Nov (rep 6.5 @ 123; mix (triticale (rep 1), 16 1); 13 Nov 2 Sep 1496; 225; 16 Apr Lancaster V ft @ 56 + ryegrass @ drilled 13 Nov Apr 2609 682 (rep 2), 24 2015 1577 239 2016 22 + clover @ 17 + 2015 2016 Nov (rep 3); radish @ 1.1); control (rep 2- 28 Nov rep 4 4) 169 31 radish @ 13; rye @ m x 101, wheat @ 101; 20 4 Oct 14 Nov Prince Georges I 6.1 mix (ryegrass @ 11 + drilled 661 108 Apr 1454 592 none none 2014 2014 m radish @ 2.5 + clover 2015 @ 5.1); control 170 Table 20. Corn yield and N response trials. Cover crop termination date, corn planting date, N fertilizer type, date applied and rates, corn herbicide information, samples taken and sampling method. V5 corn Cover Corn Corn N & Corn crops planti Date N harve Site fertilizer N rates Herbicide applied PSNT harvest terminat ng applied st type soil method ed date date sampl es Dorchester 6 Jun 8 Jun IB NA2 Crop 1 NA NA NA yes -- 2016 2016 failure3 (2016) Franklin All Silage 19 5 May IIB winter NA 0 kg ha-1 NA Unknown yes (hand Aug 2016 (2015) killed harvest) 2016 2-chloro-4-ethylamino-6-isopropylamino-s- triazine @ 0.56 kg active ing. ha-1; S-metolachor @ 0.975 kg active ing. ha-1; mesotrione @ 0.126 0 kg ha-1, kg active ing. ha-1; atrazine @ 0.975 kg active ing. Frederick Grain 12 30% 56 kg ha-1, 16 May ha-1; Simazine: 2-chloro-4,6-bis(ethylamino)-s- I NA NA -1 -1 no (harvest Oct UAN 112 kg ha , 2015 triazine @ 0.560 kg active ing. ha ; (2015) -1 er) 2015 168 kg ha Dimethylamine salt of 2,4-Dichlorophenoxyacetic acid @ 0.532 kg active ing. ha-1; glyphosate, N- (phosphonomethyl)glycine @ 2.31 kg active ing. ha-1 Frederick Silage 26 25 Apr 25 Apr IV NA NA NA NA yes (hand Aug 2016 2016 (2016) harvest) 2016 0 kg ha-1 Howard -1 12 Jun 9 May 2015; atrazine @ 1.74 kg active ing. ha -1; Grain 29 18 Apr 9 May 30% 67 kg ha IA -1 2015 2015 UAN 157 kg ha-1 2015; S-metolachor @ 1.35 kg active ing. ha ; Paraquat no (harvest Sep (2014) -1 sidedress @ 0.468 kg active ing. ha -1 er) 2015 208 kg ha Howard 30 0 kg ha-1 27 Jun 18 May 2016; atrazine @ 1.74 kg active ing. ha-1; Grain 18 18 May 30% IB May 67 kg ha-1 2016; S-metolachor @ 1.35 kg active ing. ha-1; Paraquat yes (harvest Oct 2016 UAN (2015) 2016 135 kg ha-1 sidedress @ 0.468 kg active ing. ha-1 er) 2016 171 202 kg ha-1 0 kg ha-1 Chemicals at planting; S- Metolachlor; Atrazine; 13 Kent II At corn 32% N 87 kg ha-1 10 Jun Mesotrione; May yes None NA (2015) planting UAN 174 kg ha-1 2016 Simazine; Paraquat; Lambda-cyhalothrin 2016 261 kg ha-1 (amounts NA) 50/50 S- Metolachlor @ 1.80 kg active ing. ha-1; blend of 0 kg ha-1 Atrazine @ 0.673 kg active ing. ha-1; Mesotrione Lancaster Super 84 kg ha-1 @ 0.180 kg active ing. ha-1 Grain 23 15 May 29 Apr 4 Jun 2015; IA Urea and 140 kg ha-1 no (harvest Sep 2015 2015 4 -1 sidedress (2015) Ammoni 168 kg ha er) 2015 um 224 kg ha-1 Sulfate 28% S- Metolachlor @ 1.80 kg active ing. ha-1; UAN at Atrazine @ 0.673 kg active ing. ha-1; Mesotrione planting; @ 0.180 kg active ing. ha-1 50/50 67 kg ha-1 at Lancaster 25 blend Grain 23 28 May planting; 23 Jun IB May Super 4 yes (hand Sep 2016 135 kg ha-1 2016 (2016) 2016 Urea and harvest) 2016 Ammoni um sulfate sidedress 1 Sprayed field with S-metolachor, mesotrione, atrazine (Lexar) and glyphosate, N-(phosphonomethyl)glycine (RoundUp) 2 Data not available indicated as NA 3 Crop failure due to deer damage 4 Split between planting and sidedress time 172 Table 21. Soil NO3-N and NH4-N (kg ha -1) of radish, winter cereal (cereal), mixed species (mix), and control cover crop treatments for six farms for late-fall sampling and for 11 farms for spring sampling. Different letters indicate statistically significant differences between cover crop treatments per depth increment. Farms sampled in late-fall include Dorchester IB, Frederick IV, Huntington IA, Lancaster IA, Lancaster II, and Lancaster V. Dorchester IB cores only to 180 cm deep, Frederick IV did not have soil core samples from mix treatment. Farms sampled in spring include Dorchester IB, Frederick I, Frederick III, Howard IB, Huntington IA, Lancaster IA, Lancaster IB, Lancaster II, Lancaster III, Lancaster V, and Kent II. Dorchester IB and Kent II soil cores were to only 180 cm deep. Lancaster V did not have soil core samples from mix treatment. Depth increment Radish Cereal Mix Control Radish Cereal Mix Control cm ------------ Soil NO -13-N (kg ha ) ------------- ------------ Soil NH4-N (kg ha -1) ------------- Late-fall sampling 0-30 21.8 a 22.5 a 26.8 a 69.7 b 28.6 ab 31.1 ab 36.8 a 23.0 b 30-60 13.4 a 14.1 a 22.5 a 41.6 b 14.6 a 16.9 a 18.9 a 12.7 a 60-90 13.7 a 18.0 ab 20.0 ab 29.0 b 13.1 a 15.0 a 12.0 a 10.9 a 90-120 16.2 a 19.5 a 23.8 a 24.0 a 11.9 a 14.9 a 17.6 a 11.0 a 120-150 18.8 a 22.8 a 22.4 a 25.9 a 15.0 a 21.5 a 19.8 a 15.9 a 150-180 22.1 a 20.5 a 23.7 a 23.3 a 16.7 a 19.0 a 20.9 a 17.4 a 180-210 26.8 a 21.9 a 24.3 a 25.8 a 15.0 a 19.7 a 18.7 a 15.6 a 30-90 27.1 a 32.0 a 42.5 ab 70.6 b 27.6 a 31.8 a 31.0 a 23.6 a 90-150 35.0 a 42.3 a 46.1 a 50.3 a 27.0 a 36.3 a 37.6 a 26.6 a 150-210 50.7 a 42.1 a 49.2 a 51.6 a 30.2 a 36.3 a 37.9 a 31.3 a Spring sampling 0-30 44.1 c 14.7 a 32.3 bc 31.2 b 30.6 a 23.9 a 34.3 a 30.8 a 30-60 18.6 b 4.89 a 8.7 a 19.5 b 13.6 a 10.4 a 15.8 a 14.1 a 60-90 10.7 b 4.7 a 7.98 ab 21.3 c 13.3 a 9.0 a 11.7 a 12.9 a 90-120 11.3 b 6.50 a 10.2 ab 19.7 c 11.5 a 8.6 a 11.7 a 13.2 a 120-150 17.0 b 9.6 a 13.9 ab 24.2 c 14.9 a 10.5 a 12.4 a 16.4 a 150-180 15.1 a 11.8 a 15.3 a 23.2 b 15.2 a 10.6 a 16.6 a 16.7 a 180-210 17.5 a 15.6 a 17.2 a 25.9 b 18.0 a 11.1 a 14.4 a 19.9 a 30-90 29.3 b 9.54 a 17.1 a 40.8 c 26.9 a 19.5 a 27.6 a 27.0 a 173 90-150 28.3 b 16.1 a 24.3 ab 43.9 c 26.4 a 19.2 a 24.1 a 29.6 a 150-210 29.7 a 25.0 a 30.0 a 45.4 b 30.4 a 19.1 a 27.6 a 33.4 a 174 Table 22. Fall and spring sum of NO3-N (kg ha -1) and NH -14-N (kg ha ) from 0-90 cm and from 0-210 cm deep for 14 farms, and cover crop biomass (kg ha-1), N content (kg N ha-1), and C/N ratio for 19 farms. Cover crop treatment values for a response variable, within the same season and farm, followed by the different letters are significantly different (p <0.05); * indicates significantly different (p < 0.1). Cover crop Soil Late-fall sampling Spring sampling Late-fall sampling Spring sampling NO3-N NH4-N NO3-N NH4-N Bioma N C/N Bioma N C/N ss content ratio ss content ratio 0-90 90-210 0-90 90-210 0-90 90-210 0-90 90-210 cm cm cm cm cm cm cm cm kg ha-1 6298 14.1 Radish 82.4 a 27.2 a NA NA NA 18.6 a 24.0 a 86.5 a 84.8 a 14.2 a 32.0 a 25.9 a a ab 1529 5.93 Dorches Wheat 29.2 b 21.1 b 814 a 10.8 a 22.5 a 16.2 a 23.8 a 62.7 a 61.5 a 8.63 a 40.3 a 29.9 a b ab ter IB1 4509 1047 Mix 61.0 c 27.5 a 12.0 a 28.0 a 12.3 a 24.0 a 63.3 a 77.5 a 3.40 a 16.2 a 28.9 a 20.7 a c a Contro -- -- -- -- -- -- 22.8 a 21.1 a 43.2 a 51.4 a 23.0 b 12.1 a 35.3 a 25.5 a l 2829 46.0 Radish 48.1 a 22.8 a NA NA NA -- -- -- -- 44.9 a 66.0 a 94.9 a a ab Tritica 1194 1263 23.8 b 21.1 a 26.2 a 18.7 b -- -- -- -- 26.9 a 50.6 a 58.4 a 57.1 a Frederic le b a k I Contro 2371 1210 47.8 a 19.7 a 28.0 a 15.7 a 36.2 a 49.9 a 31.5 a 22.6 a 31.1 a 38.7 a 57.0 a 54.6 a l c a Contro -- -- -- -- -- -- 58.2 a 63.9 a 25.9 a 21 a 60.3 b 58.7 a 99.5 a 118 a l 31.9 Radish -- -- -- -- -- -- -- -- -- -- 93.0 a 84.4 a 122 a ab Frederic Tritica 1183 k III -- -- -- 30.3 a 15.9 a -- -- -- -- 14.5 a 90.1 a 43.2 a 64.5 a le a 1079 Mix -- -- -- 30.3 a 13.3 b -- -- -- -- 19.2 a 116 a 39.4 a 48.7 a a 175 Contro 100 -- -- -- -- -- -- -- -- -- -- 57.9 b 161 a 170 a l a 2182 Radish 53.9 a 15.2 a NA NA NA 35.1 a 39.1 a 83.7 a 56.5 a -- -- -- -- a Frederic Tritica 1140 k IV2 31.3 a 15.1 a 4799 55.5 36.8 40.0 a 69.4 b* 94.3 a 128 a -- -- -- -- le b Contro -- -- -- -- -- -- 84.6 b 47 ab 98.7 a 119 a -- -- -- -- l 3477 Radish 79.5 a 18.1 a -- -- -- 36.6 a 149 a 43.0 a 30.5 a -- -- -- -- a Harford 1967 Mix 50.5 a 16.2 a -- -- -- 38.6 a 69.7 a 40.6 a 27 a -- -- -- -- I a Contro -- -- -- -- -- -- 85.0 b 76.1 a 42.0 a 26.8 a -- -- -- -- l 2269 Radish 45.9 a 19.1 a NA NA NA 31.3 a 14.6 a 31.4 a 5.05 a -- -- -- -- a Howard 1158 IA3 Rye 28.9 b 16.8 b 1345 29 19.5 -- -- -- -- -- -- -- -- b Contro -- -- -- -- -- -- 77.3 a 10.1 a 30.0 a 9.15 a -- -- -- -- l 1964 140 Radish 46.3 a 13.1 a NA NA NA -- -- -- -- 56.4 c 50.3 a 173 a a a 1150 3165 Rye 26.9 b 16.5 b 41.2 a 30.5 b -- -- -- -- 16.1 a 37.7 a 70.7 a 85.0 a Howard b a IB 2095 13.7 3571 27.7 143 Mix 56.1 a 68.2 b 21.7 a -- -- -- -- 52.4 a 188 a a ab a ab a Contro -- -- -- -- -- -- -- -- -- -- 38.8 b 55.9 a 83.3 a 94.6 a l 3717 163 Radish 122 a 12.0 a NA NA NA 59.6 a 208 a 62.0 a 86 a 172 ab 62.7 a 38.4 a a b Huntingt 2315 104 132 on IA Rye 83.2 a 12.1 a 4533 126 14.7 166 a 137 a 39.9 a 110 a* 34.7 a 26.2 a b a a 3864 146 225 Mix 132 a 11.7 a NA NA NA 234 a 81.9 a 88.7 a 165 ab 68.6 a 47.3 a a a c 176 Contro 178 194 -- -- -- -- -- -- 254 a 63.4 a 80.3 a 292 b 44.0 a 62.9 a l a bc 2380 Radish 37.3 a 23.4 a NA NA NA -- -- -- -- 41.5 a 5.43 a 21.0 a 4.9 a a 1055 1325 Rye 20.5 b 20.1 b 15.8 a 34.9 a -- -- -- -- 22.1 a 6.85 a 27.0 a 7.73 a b a 2039 21.0 2271 Kent II4 Mix 36. 6 a 25.4 a 37.3 a -- -- -- -- 12.8 a 7.00 a 92.0 a 46.0 a ab ab b Late- 1216 2244 26.5 ab 16.4 c 27.7 a 34.1 a -- -- -- -- 33.9 a 20.5 a 76.5 a 50.6 a mix b b Contro -- -- -- -- -- -- -- -- -- -- 28.9 a 23.3 a 21.5 a 12.9 a l 99.8 77.4 Radish -- -- -- -- -- -- 82.3 a 61.9 a 56.7 a 21.9 a 30.8 a 17.5 a ab ab Tritica 60.5 Lancaste 4333 115 15.8 3988 87.5 18.9 67.0 a 36.6 a 30.9 a 20.9 a 8.43 a 23.8 a 14.6 a le a* r IA5 92.2 73.0 Mix -- -- -- -- -- -- 81.2 a 61.9 a 69.4 a 19.4 a 34.9 a 34.8 ab ab ab Contro 129 126 -- -- -- -- -- -- 80.8 a 31.9 a 18.5 a 83.4 b 38.0 a 37.3 b* l b b 4162 Radish 86.9 a 17.3 a NA NA NA -- -- -- -- 72.7 a 34.0 a 80.6 a 37.3 a a Tritica 1291 13.1 7321 16.8 40.1 b 60.0 a 52.4 b -- -- -- -- 36.0 a 50.3 a 42.2 a le b bc a b* Lancaste 2240 54. 7 15.7 7459 35.3 r IB Mix 88.9 a 36.8 a -- -- -- -- 25.6 a 92.9 a 33.5 a b ab ab a ab Late- 1249 9479 26.3 38.54 b 11.1 c 174 a 28.1 a -- -- -- -- 36.2 a 62.2 a 16.5 a mix b a ab Contro -- -- -- -- -- -- -- -- -- -- 67.8 a 74.9 b 79.5 a 31.1 a l 5712 58.0 Radish 202 a 11.1 a NA NA NA 33.1 a 58.8 a 20.3 a 10.9 a 42.9 ab 18.1 a 14.2 a Lancaste a ab r II Tritica 3607 3014 126 a 12.1 a 88.8 a 14.0 b 36.5 a 76.2 a 27.1 a 31.8 b 24.8 a 23.8 a 22.8 a 9.87 a le a a 177 4900 1503 47.8 Mix 165 a 12.2 a 51.4 b 11.8 a 51.1 a 67.9 a 47.7 b 37.1 b 36.8 ab 19.4 a 13.0 a a b ab Contro 125 -- -- -- -- -- -- 54.8 a 16.6 a 10.7 a 79.9 b 54.3 b 23.8 a 13.1 a l b Radish -- -- -- -- -- -- -- -- -- -- 64.1 a 31.7 a 49.4 a 40.8 a Tritica 2619 Lancaste -- -- -- 53.9 a 20.4 b -- -- -- -- 36.2 b 17.0 b 62.6 a 29.7 a le a r III6 2572 Mix -- -- -- 67.1 a 16.1 a -- -- -- -- 30.1 b 16.0 b 45.4 a 25.9 a a Contro 54.4 -- -- -- -- -- -- -- -- -- -- 30.5 a 63.5 a 44.3 a l ab 4915 183 Radish 186 a 8.50 b NA NA NA 55.2 a 60.6 a 33.5 a 6.83 a 111 a 26.3 a 11.7 a a a Tritica 3448 9.14 7125 Lancaste 148 b 202 a 14.0 b 74.2 a 71.5 a 35.7 a 8.28 a 44.0 b 55.1 b 33.2 a 13.3 a le b ab a r V 5326 1830 Mix 204 a 9.4 a 68.3 b 10.6 a 70.0 a 76.1 a 60.7 a 31.3 a -- -- -- -- a b Contro 280 -- -- -- -- -- -- 116 b* 25.6 a 8.1 a 81.7 b 127 a 24.6 a 19.0 a l b 1330 Radish 48.0 a 10.8 a NA NA NA -- -- -- -- -- -- -- -- a Franklin Tritica 2620 837 a 29.2 a 12.0 a 68.4 a 15.5 b -- -- -- -- -- -- -- -- I le a 1359 1653 Mix 42.0 a 13.2 a 48.8 a 13.9 a -- -- -- -- -- -- -- -- a a 3359 Radish 117 a 10.9 a NA NA NA -- -- -- -- -- -- -- -- a Franklin 1848 Oat 57.4 b 14.9 a NA NA NA -- -- -- -- -- -- -- -- IIA b 2853 Mix 82.5 ab 14.5 a NA NA NA -- -- -- -- -- -- -- -- ab Radish 900 a 31.0 a 10.2 a NA NA NA -- -- -- -- -- -- -- -- 178 Franklin Oat 569 b 23.1 a 9.3 a NA NA NA -- -- -- -- -- -- -- -- IIB Mix 977 a 38.3 a 9.8 a NA NA NA -- -- -- -- -- -- -- -- 3023 Radish -- -- NA NA NA -- -- -- -- -- -- -- -- a Huntingt Ryegr 1567 -- -- -- -- -- -- -- -- -- -- -- -- -- on IB ass a 2240 Mix -- -- -- -- -- -- -- -- -- -- -- -- -- a Radish 222 a 7.76 a 10.3 b NA NA NA -- -- -- -- -- -- -- -- Prince Rye 146 a 4.85 ab 12.9 a 826 a 15.0 a 23.0 a -- -- -- -- -- -- -- -- Georges 1024 Wheat -- -- -- 17.0 a 25.5 a -- -- -- -- -- -- -- -- I a 11.1 Mix 128 a 3.53 b 921 a 14.3 a 26.1 a -- -- -- -- -- -- -- -- ab 1 Dorchester IB soil was sampled only to 180 cm for fall and spring samples. 2 Frederick IV soil was sampled only to 180 cm in fall. 3 Howard IA soil was sampled only to 120 cm in fall. 4 Kent II soil was sampled only to 180 cm in spring. 5 Lancaster IA soil was sampled only to 180 cm in spring. 6 Lancaster III soil was sampled only to 180 cm in spring. 179 Table 23. Cover crop biomass (kg ha-1), N content (kg N ha-1), and C/N ratio for fall cover crop growth (late-fall sampling), fall and spring cover crop growth (spring sampling), and cover crop growth prior to termination (late-fall radish, prior to winter- kill, and spring winter cereal, prior to herbicide termination. Different letters indicate statistically significant differences between cover crop treatments. Cover crop treatment N Biomass (kg ha-1) N content (kg ha- C/N ratio 1) Late-fall sampling1 Radish 13 3085 a 85.5 a 14.2 a Winter cereal 13 1586 c 52.1 b 14.2 a Mixed species 13 2651 b 77.6 a 14.5 a Spring sampling2 Winter cereal 10 3046 b 61.3 a 23.2 b Mixed species 10 2385 a 49.9 a 19.2 a Sampling prior to termination3 Radish before termination 13 2663 a 79.7 a 13.4 a Winter cereal before 13 3026 a 61.6 b 22.6 b termination 1 Values reported for late-fall sampling from 13 farms—Franklin I, Howard IB, Lancaster II, Prince Georges I, Lancaster V, Franklin IIA, Franklin IIB, Lancaster IB, Huntington IA, Huntington IB, Dorchester IB, Frederick I, Kent II. 2 Values reported for spring sampling from 10 farms—Franklin I, Howard IB, Lancaster II, Prince Georges I, Lancaster V, Lancaster IB, Frederick III, Lancaster III, Frederick I, Kent II. 3 Values reported for sampling prior to termination from 13 farms—Franklin I, Franklin IIA, Franklin IIB, Frederick I, Frederick IV, Howard IA, Howard IB, Huntington IA, Kent II, Lancaster IB, Lancaster II, Lancaster V, and Prince Georges I). On Franklin IIA and Franklin IIB, the winter cereal (oat) naturally winter-killed rather than being chemically terminated. 180 Table 24. Correlations among cover crop biomass and growing degree days (GDD), precipitation (prec.) total from cover crop planting date to sampling date, topsoil (0-30 cm) NO3-N and NH4-N (kg N ha -1), topsoil percent sand/clay/silt. Table showing number of replicates (N), corr elation coefficient (r), and p-value of correlation. Biomass GDD Prec. NO3-N NH4-N % sand % clay % silt Fall winter cereal N 16 16 14 14 14 14 14 r 0.288 0.184 0.774 0.039 -0.521 0.224 0.517 p-value 0.28 0.494 0.001 0.895 0.056 0.442 0.058 Fall radish N 16 16 14 14 14 14 14 r 0.433 0.233 0.484 0.118 -0.467 -0.011 0.558 p-value 0.094 0.385 0.080 0.688 0.092 0.970 0.038 Spring winter cereal N 15 15 15 15 15 15 15 r 0.629 0.576 0.400 -0.271 -0.744 0.438 0.703 p-value 0.012 0.025 0.140 0.328 0.002 0.102 0.004 181 Table 25. June PSNT soil sample NO3-N and NH4-N concentrations (mg N kg -1 soil), and corn plant biomass per corn plant (g plant-1) and N in biomass per corn plant (g N plant-1) following cover crop treatments. N indicates the number of replicates per cover crop treatment. Cover crop treatment values for a response variable, within the same farm, followed by the different letters are significantly different (p < 0.05). Site N RADISH CEREAL MIX Late-mix CONTROL NO3-N 3 10.56 a 14.15 a 5.80 a -- 10.4a Dorchester IB NH4-N 3 5.39 a 7.37 a 7.38 a -- 7.80 a Corn biomass 3 1.19 a 0.609 b 0.862 ab -- 1.10 ab Corn N 3 0.0531 a 0.0283 b 0.0395 ab -- 0.0479 ab NO3-N 4 6.53 a 7.02 a 8.03 a -- 5.11 a Franklin IIB NH4-N 4 6.77 a 7.11 a 4.98 a -- 5.72 a Corn biomass 4 3.25 a 2.67 a 2.38 a -- 2.41 a Corn N 4 0.114 a 0.0965 a 0.0904 a -- 0.0894 a NO3-N 4 13.11 a 7.32 a -- -- 11.43 a Frederick IV NH4-N 4 0.923 a 1.124 a -- -- 0.724 a Corn biomass 4 3.15 a 1.50 b -- -- 3.23 a Corn N 4 0.115 a 0.0557 b -- -- 0.116 a NO3-N 4 5.37 a 2.09 b 3.46 ab -- 3.30 ab Howard IB NH4-N 4 5.19 a 7.16 a 7.24 a -- 10.36 a Corn biomass 4 7.00 a 3.32 c 5.72 b -- 5.38 b Corn N 4 0.234 a 0.090 c 0.172 b -- 0.158 b NO3-N 3 8.00 a 4.66 ab 3.56 b 3.78 b 6.46 ab Kent II NH4-N 3 8.15 a 7.16 a 7.93 a 6.44 a 8.11 a Corn biomass 3 3.01 a 2.13 a 2.30 a 2.51 a 2.50 a Corn N 3 0.129 a 0.0781 a 0.0887 a 0.0997 a 0.102 a NO3-N 4 19.4 a 7.24 a 10.6 a 11.5 a 11.7 a Lancaster IB NH4-N 4 10.2 a 9.20 a 11.09 a 9.71 a 9.22 a Corn biomass 4 5.58 a 3.51 b 3.78 b 4.38 ab 5.29 a Corn N 4 0.246 a 0.155 b 0.175 b1 0.201 ab 0.236 a NO3-N 10.6 a 6.87 b 6.79 b -- 8.04 ab 6 Farms NH4-N 6.04 a 6.45 a 6.64 a -- 6.90 a Corn biomass 84 4.02 a 2.37 c 3.03 b -- 3.46 b2 Corn N 84 0.154 a 0.0867 c 0.113 b -- 0.129 b 1 Difference between a and b significant at p < 0.055. 2 Difference between a and b significant at p < 0.056. 182 Table 26. Percent of maximum corn yield following cover crop treatments for farmers’ standard fertilizer application rate (standard) or no fertilizer application. Cover crop treatment values for percent of maximum corn yield, within the same fertilizer N level, followed by different letters are significantly different (p < 0.05). Site N rate, kg ha-1 Radish Winter cereal Mix Control Percent of maximum yield 6 farms1 Standard2 92% a 71% c 77% bc 86% ab 6 farms3 0 fertilizer4 85% a 56% c 73% b 73% b 1 Farms include Frederick IV (corn silage) and Howard IA, Howard IB, Frederick I, Lancaster IA and Lancaster IB (corn grain) 2 Rate that farmer normally uses 3 Farms include Franklin IIB (corn silage) and Howard IA, Howard IB, Frederick I, Lancaster IA and Lancaster IB (corn grain) 4 Lancaster IB applied 67 kg N ha-1 at planting 183 150 100 50 0 -50 -100 -150 -200 -250 -300 -350 -400 -450 -500 -550 -600 fall spring fall spring spring fall fall fall spring fall spring spring Dorchester IB Frederick I Frederick III Frederick Harford I Howard IA Huntington IA Kent II IV CC N NO3-N 0-90 cm NO3-N 90-210 cm Figure 15. Cover crop N uptake (CC N; green bars) and soil NO -13-N (kg ha ) from 0-90 cm (brown bars) and 90-210 cm (orange bars) for 13 farms with biomass and soil samples collected. Belowground N (soil) indicated with negative values. Standard error bars show. 184 Cover crop N or soil NO3-N (kg ha -1) radish wheat mix control radish wheat mix control mix control radish triticale mix control radish triticale mix control radish triticale control radish mix control radish control radish rye mix control radish rye mix control radish rye mix control radish rye early-mix late-mix control 250 200 150 100 50 0 -50 -100 -150 -200 -250 -300 -350 -400 -450 fall spring spring fall spring spring fall spring Lancaster IA Lancaster IB Lancaster II Lancaster III Lancaster V CC N NO3-N 0-90 cm NO3-N 90-210 cm Figure 15 continued 185 Cover crop N or soil NO3-N (kg ha -1) triticale control radish triticale control radish rye early-mix late-mix control radish triticale mix control radish triticale mix control radish triticale mix control radish triticale mix control radish triticale control Figure 16. Amount of NO3-N (kg ha -1) in 0-210 cm soil profile for seven farms at fall sampling and 10 farms at spring sampling. 1Spring samples from 120-150 cm and 150-180 cm depths are the average values from 120-180 cm. 186 Figure 16 continued 187 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 30 35 June 0-30 cm soil NO -13-N (mg kg ) Control Mix Radish Winter cereal Figure 17. Relationship between fraction of maximum corn yield (no N applied on corn) and pre-sidedress test nitrate concentrations for cover crop treatments. Data from Howard IB, Franklin IIB, and Lancaster IB. 188 Fraction of maximum corn yield 4.5 a* 4.0 b 3.5 b 3.0 c 2.5 2.0 1.5 1.0 0.5 0.0 Control Winter cereal Mix Radish Previous cover crop treatment Figure 18. Corn biomass at V5 growth stage for cover crop treatments. Corn biomass values with different letters are significantly different (p < 0.05). Data from Dorchester IB, Frederick IV, Howard IB, Franklin IIB, Kent II and Lancaster IB. *Differences between radish and control at p < 0.0561. 189 V5 stage corn plant biomass (g plant-1) 14000 14000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 Howard IA 2000 Howard IB 0 0 0 67 157 208 0 67 135 202 N fertilizer applied (kg N ha-1) Fertilizer N applied (kg N ha-1) 12000 16000 14000 10000 12000 8000 10000 6000 8000 6000 4000 4000 2000 Frederick I 2000 Lancaster IA 0 0 0 56 112 168 0 84 140 168 224 N fertilizer applied (kg N ha-1) N fertilizer applied (kg N ha -1) 10000 Control 8000 Mix 6000 Late-mix Radish 4000 Lancaster IB Winter cereal 2000 0 67 135 N fertilizer applied (kg N ha-1) Figure 19. Corn grain yield associated with various N fertilizer rates and preceding cover crop treatments. Error bars show standard error of mean. 190 Grain yield (kg ha-1) Grain yield (kg ha-1) Grain yield (kg ha-1) Appendix 7. Supplemental soil characteristics of study sites Table 27. Study site soil pH, percent sand, percent clay, percent C, and percent N for each 15 or 30 cm soil depth increment (0-210 cm), and percent soil organic matter (SOM), P, K, Mg, Ca, and S (mg kg-1) for the upper 30 cm of soil from each farm site. Each record is the average of two to three composited soil cores from two areas in the field. Data from Dorchester IB 180-210 cm and Lancaster IB 180-210 cm is from a single point of a field. Soil samples were not taken from Franklin IIA or Huntington IB; however these sites were within 100 meters of Franklin IIB and Huntington IA, respectively. Values not determined indicated as nd. Values below detection limit indicated as BDL. Farm Depth pH1 Sand2 Clay2 C3 N3 SOM4 P4 K4 Mg4 Ca4 S4 site cm % ppm 75. 0-30 6.0 34.9 12.8 0.805 0.071 2.25 47.5 43 643 20 5 30-60 5.2 35.4 20 0.17 0.022 -- -- -- -- -- -- Dorch 60-90 4.5 20.1 19.6 0.16 0.027 -- -- -- -- -- -- ester 90-120 4.8 45.7 17.6 0.15 0.025 -- -- -- -- -- -- IB 120-150 4.9 59.2 15.3 0.146 BDL -- -- -- -- -- -- 150-180 5.1 62.9 15.2 0.13 0.020 -- -- -- -- -- -- 180-210 6.0 27.1 26.9 0.181 0.033 -- -- -- -- -- -- 0-15 5.5 45.9 18.9 0.943 0.092 2.35 19 69 127 777 18.5 15-30 5.9 46.5 22.4 0.458 0.051 1.65 9.5 56 122 718 15.5 30-45 nd nd nd 0.309 0.037 -- -- -- -- -- -- 45-60 5.8 39 32.7 0.393 0.046 -- -- -- -- -- -- 60-75 nd nd nd 0.183 0.028 -- -- -- -- -- -- 75-90 6.5 29.3 40.6 0.153 0.028 -- -- -- -- -- -- Frankl 90-105 nd nd nd 0.232 0.034 -- -- -- -- -- -- in I 105-120 5.8 29.2 40.5 0.177 0.028 -- -- -- -- -- -- 120-135 nd nd nd 0.211 0.031 -- -- -- -- -- -- 135-150 4.8 26.7 27.9 0.22 0.031 -- -- -- -- -- -- 150-165 nd nd nd 0.202 0.032 -- -- -- -- -- -- 165-180 4.9 21.7 29.4 0.118 0.026 -- -- -- -- -- -- 180-195 nd nd nd 0.131 0.027 -- -- -- -- -- -- 195-210 4.9 28.3 26.2 0.152 0.025 -- -- -- -- -- -- 80. 142 0-30 6.8 17.9 22 1.258 0.120 2.9 49 111 3.5 5 1 30-60 6.7 11.4 33.2 0.588 0.064 -- -- -- -- -- -- Frankl in IIB 60-90 6.6 18.4 30.9 0.441 0.049 -- -- -- -- -- -- 90-120 5.9 30.6 47.8 0.303 0.044 -- -- -- -- -- -- 120-150 5.6 27.9 59.9 0.179 0.048 -- -- -- -- -- -- 191 150-180 5.1 17.6 54 0.141 0.043 -- -- -- -- -- -- 180-210 5.0 13.9 56.8 0.152 0.045 -- -- -- -- -- -- 71. 0-15 6.4 38.5 16.5 1.097 0.118 2.75 80 125 948 12 5 15-30 6.2 41.1 17.4 0.536 0.060 1.85 24.5 48 101 929 12.5 30-45 nd nd nd 0.319 0.046 -- -- -- -- -- -- 45-60 6.2 36.3 28.6 0.224 0.036 -- -- -- -- -- -- 60-75 nd nd nd 0.15 0.033 -- -- -- -- -- -- 75-90 5.2 32 30.9 0.119 0.024 -- -- -- -- -- -- Freder 90-105 nd nd nd 0.13 0.027 -- -- -- -- -- -- ick I 105-120 4.9 42.7 24.1 0.131 0.030 -- -- -- -- -- -- 120-135 nd nd nd 0.183 0.034 -- -- -- -- -- -- 135-150 4.8 50.1 11.3 0.082 BDL -- -- -- -- -- -- 150-165 nd nd nd 0.077 BDL -- -- -- -- -- -- 165-180 4.7 47.2 11.4 0.064 0.020 -- -- -- -- -- -- 180-195 nd nd nd 0.081 0.023 -- -- -- -- -- -- 195-210 4.9 49.8 10.4 0.082 BDL -- -- -- -- -- -- 180 0-15 6.9 29.1 24.7 1.498 0.181 4.3 87.5 83 93 15 2 97. 137 15-30 6.7 26.8 32.3 0.606 0.102 2.4 20 47 8.5 5 5 30-45 nd nd nd 0.258 0.079 -- -- -- -- -- -- 45-60 6.5 23.9 37.4 0.202 0.077 -- -- -- -- -- -- 60-75 nd nd nd 0.142 0.075 -- -- -- -- -- -- 75-90 6.0 19.7 35.5 0.116 0.073 -- -- -- -- -- -- Freder 90-105 nd nd nd 0.094 0.068 -- -- -- -- -- -- ick III 105-120 5.2 18.5 34.3 0.086 0.063 -- -- -- -- -- -- 120-135 nd nd nd 0.174 0.077 -- -- -- -- -- -- 135-150 4.9 23.8 29 0.082 0.067 -- -- -- -- -- -- 150-165 nd nd nd 0.077 0.069 -- -- -- -- -- -- 165-180 4.9 24.4 30.1 0.072 0.061 -- -- -- -- -- -- 180-195 nd nd nd 0.079 0.065 -- -- -- -- -- -- 195-210 4.6 30.7 26.2 0.071 0.061 -- -- -- -- -- -- 73. 147 0-30 7.3 29.1 22.5 1.025 0.105 2.2 69.5 108 3.5 5 0 30-60 7.4 18.7 40 0.285 0.041 -- -- -- -- -- -- 60-90 7.4 18.5 48.5 0.223 0.043 -- -- -- -- -- -- Freder ick IV 90-120 6.6 21.6 46.2 0.163 0.038 -- -- -- -- -- -- 120-150 6.7 28.3 38.7 0.137 0.033 -- -- -- -- -- -- 150-180 7.1 26.5 45.9 0.169 0.044 -- -- -- -- -- -- 180-210 7.7 28.5 36.7 1.557 0.039 -- -- -- -- -- -- 192 0-15 6.1 26.6 19.5 1.738 0.159 4.3 17 95 133 728 11 15-30 5.7 24.9 25.6 0.76 0.078 2.3 4 59 99 543 29 30-45 nd nd nd 0.433 0.051 -- -- -- -- -- -- 45-60 5.3 29.5 23.3 0.204 0.032 -- -- -- -- -- -- 60-75 nd nd nd 0.157 0.024 -- -- -- -- -- -- 75-90 5.1 38.5 16.9 0.107 BDL -- -- -- -- -- -- Harfor 90-105 nd nd nd 0.083 BDL -- -- -- -- -- -- d I 105-120 5.2 36.3 14.7 0.099 0.020 -- -- -- -- -- -- 120-135 nd nd nd 0.258 0.032 -- -- -- -- -- -- 135-150 5.2 40.5 9.9 0.118 0.026 -- -- -- -- -- -- 150-165 nd nd nd 0.093 BDL -- -- -- -- -- -- 165-180 5.2 44.2 8.1 0.1 0.022 -- -- -- -- -- -- 180-195 nd nd nd 0.056 BDL -- -- -- -- -- -- 195-210 5.1 43.2 13.2 0.071 0.021 -- -- -- -- -- -- 170 0-15 6.5 43.9 12.6 2.193 0.214 4.2 60.5 126 111 17.5 5 59. 146 15-30 6.5 47.7 15.7 1.126 0.103 2.6 36.5 104 16.5 5 4 30-45 nd nd nd 1.297 0.108 -- -- -- -- -- -- 45-60 6.8 25.5 32.6 1.423 0.111 -- -- -- -- -- -- 60-75 nd nd nd 1.324 0.097 -- -- -- -- -- -- 75-90 6.9 35.3 27.6 0.883 0.062 -- -- -- -- -- -- Howa 90-105 nd nd nd 0.682 0.047 -- -- -- -- -- -- rd IA 105-120 6.6 35.9 10.2 0.724 0.050 -- -- -- -- -- -- 120-135 nd nd nd 0.758 0.058 -- -- -- -- -- -- 135-150 6.8 47.7 24.9 0.438 0.030 -- -- -- -- -- -- 150-165 nd nd nd 0.374 0.028 -- -- -- -- -- -- 165-180 6.7 48.3 18.6 0.363 0.023 -- -- -- -- -- -- 180-195 nd nd nd 0.229 0.020 -- -- -- -- -- -- 195-210 7.0 68.9 12.3 0.256 BDL -- -- -- -- -- -- 51. 0-30 6.2 47.8 16.7 0.703 0.071 2.1 16.5 76 571 16 5 30-60 6.5 50.7 17.2 0.484 0.048 -- -- -- -- -- -- 60-90 6.4 29.2 23.8 0.364 0.039 -- -- -- -- -- -- Howa rd IB 90-120 6.1 23.7 28.3 0.207 0.034 -- -- -- -- -- -- 120-150 5.7 30.5 23.9 0.134 0.027 -- -- -- -- -- -- 150-180 5.9 42 19.8 0.139 0.023 -- -- -- -- -- -- 180-210 5.9 39.9 19.3 0.157 0.021 -- -- -- -- -- -- Hunti 1370-15 6.5 27 20.6 2.829 0.228 5.4 93.5 228 277 8.5 ngton 8 IA 15-30 6.0 24.3 25.5 0.792 0.076 2.6 23.5 127 164 680 22.5 193 30-45 nd nd nd 0.369 0.050 -- -- -- -- -- -- 45-60 4.8 19.2 38 0.213 0.038 -- -- -- -- -- -- 60-75 nd nd nd 0.163 0.033 -- -- -- -- -- -- 75-90 4.7 22.7 35.1 0.155 0.033 -- -- -- -- -- -- 90-105 nd nd nd 0.217 0.034 -- -- -- -- -- -- 105-120 4.6 39.7 28.3 0.117 0.027 -- -- -- -- -- -- 120-135 nd nd nd 0.668 0.062 -- -- -- -- -- -- 135-150 4.7 18.9 46.3 0.146 0.039 -- -- -- -- -- -- 150-165 nd nd nd 0.178 0.029 -- -- -- -- -- -- 165-180 4.6 28.1 46 0.13 0.033 -- -- -- -- -- -- 180-195 nd nd nd 0.099 0.034 -- -- -- -- -- -- 195-210 4.5 16.3 49.4 0.174 0.038 -- -- -- -- -- -- 0-15 6.0 45.8 8.2 1.015 0.101 2.65 27.5 72 58 495 2 15-30 5.7 44.5 10.1 0.509 0.056 -- -- -- -- -- -- 30-45 nd nd nd 0.292 0.036 -- -- -- -- -- -- 45-60 5.9 36.3 17.9 0.222 0.038 -- -- -- -- -- -- 60-75 nd nd nd 0.167 0.033 -- -- -- -- -- -- 75-90 5.8 49.3 17.7 0.12 0.027 -- -- -- -- -- -- Kent 90-105 nd nd nd 0.067 0.022 -- -- -- -- -- -- IIB 105-120 5.6 85 5.6 0.052 0.015 -- -- -- -- -- -- 120-135 nd nd nd 0.268 0.030 -- -- -- -- -- -- 135-150 5.1 84 3.5 0.052 BDL -- -- -- -- -- -- 150-165 nd nd nd 0.05 BDL -- -- -- -- -- -- 165-180 5.3 91.4 3.7 0.038 BDL -- -- -- -- -- -- 180-195 nd nd nd 0.031 BDL -- -- -- -- -- -- 195-210 4.8 92.6 2.7 0.035 BDL -- -- -- -- -- -- 140 0-15 6.7 29.2 20.6 1.952 0.202 4.7 87 154 116 29.5 1 47. 15-30 6.3 33.1 22.6 0.953 0.098 3.1 33.5 84 841 7.5 5 30-45 nd nd nd 0.286 0.039 -- -- -- -- -- -- 45-60 6.4 35.3 27.3 0.414 0.049 -- -- -- -- -- -- Lanca 60-75 nd nd nd 0.176 0.029 -- -- -- -- -- -- ster 75-90 6.4 44.9 23.3 0.09 BDL -- -- -- -- -- -- IA 90-105 nd nd nd 0.102 BDL -- -- -- -- -- -- 105-120 6.3 52.8 18.2 0.079 BDL -- -- -- -- -- -- 120-135 nd nd nd 0.266 0.038 -- -- -- -- -- -- 135-150 5.9 55.4 17.6 0.095 0.022 -- -- -- -- -- -- 150-165 nd nd nd 0.078 BDL -- -- -- -- -- -- 194 165-180 5.5 55.5 17.2 0.06 BDL -- -- -- -- -- -- 180-195 nd nd nd 0.061 BDL -- -- -- -- -- -- 195-210 5.4 62.5 11 0.044 BDL -- -- -- -- -- -- 0-30 6.1 30.5 25.2 0.869 0.087 3.45 81.5 38 60 763 13.5 30-60 6.2 38.6 29.9 0.292 0.034 -- -- -- -- -- -- 60-90 6.1 51.5 26.5 0.167 0.021 -- -- -- -- -- -- Lanca ster 90-120 5.7 55.1 18.4 0.095 BDL -- -- -- -- -- -- IB 120-150 6.1 62.3 15.6 0.14 BDL -- -- -- -- -- -- 150-180 6.0 67.4 8.3 0.084 BDL -- -- -- -- -- -- 180-210 5.7 73.7 4.4 0.054 BDL -- -- -- -- -- -- 159 0-15 6.7 32.2 16.5 1.654 0.184 3.95 104 120 130 45.5 9 81. 135 15-30 6.8 31 21.3 1.035 0.123 3.1 54.5 107 37.5 5 6 30-45 nd nd nd 0.331 0.053 -- -- -- -- -- -- 45-60 6.7 39 16.9 0.264 0.048 -- -- -- -- -- -- 60-75 nd nd nd 0.094 0.029 -- -- -- -- -- -- 75-90 6.8 50.7 7 0.075 0.026 -- -- -- -- -- -- Lanca 90-105 nd nd nd 0.056 0.023 -- -- -- -- -- -- ster II 105-120 6.8 53.7 3.1 0.036 BDL -- -- -- -- -- -- 120-135 nd nd nd 0.153 0.036 -- -- -- -- -- -- 135-150 6.8 60.1 2.3 0.037 0.025 -- -- -- -- -- -- 150-165 nd nd nd 0.029 0.023 -- -- -- -- -- -- 165-180 6.7 63.4 2.3 0.027 0.027 -- -- -- -- -- -- 180-195 nd nd nd 0.028 0.025 -- -- -- -- -- -- 195-210 6.9 57.2 2.7 0.029 0.023 -- -- -- -- -- -- 118 0-15 6.4 46.6 12.7 1.484 0.164 3.15 101 185 207 2 7 15-30 6.5 49 14.8 0.847 0.096 2.1 59.5 137 155 940 4 30-45 nd nd nd 0.337 0.046 -- -- -- -- -- -- 45-60 5.8 47.6 16 0.519 0.060 -- -- -- -- -- -- 60-75 nd nd nd 0.52 0.060 -- -- -- -- -- -- Lanca 75-90 5.9 45.7 18.7 0.335 0.045 -- -- -- -- -- -- ster 90-105 nd nd nd 0.267 0.040 -- -- -- -- -- -- III 105-120 5.2 43.9 17.9 0.158 0.028 -- -- -- -- -- -- 120-135 nd nd nd 0.204 0.032 -- -- -- -- -- -- 135-150 4.9 51 17.9 0.114 0.022 -- -- -- -- -- -- 150-165 nd nd nd 0.084 0.020 -- -- -- -- -- -- 165-180 5.3 50.3 17.3 0.095 0.020 -- -- -- -- -- -- 180-195 nd nd nd 0.113 0.021 -- -- -- -- -- -- 195 195-210 5.4 44.9 15.4 0.116 BDL -- -- -- -- -- -- 162 0-30 7.1 10.7 17.1 1.581 0.153 2.75 120 134 196 12.5 6 30-60 6.8 14 24.4 0.56 0.055 -- -- -- -- -- -- 60-90 7.0 13.9 23.3 0.352 0.034 -- -- -- -- -- -- Lanca ster V 90-120 7.1 17.2 23.6 0.223 0.025 -- -- -- -- -- -- 120-150 7.0 30.9 22 0.186 0.021 -- -- -- -- -- -- 150-180 6.8 26.2 26.4 0.132 BDL -- -- -- -- -- -- 180-210 7.1 24.7 32 0.254 0.030 -- -- -- -- -- -- 0-15 5.2 59.7 11.9 1.416 0.128 2.9 61 144 165 633 14 15-30 5.2 56.5 16 0.657 0.068 1.85 42 112 152 643 9.5 30-45 nd nd nd 0.55 0.063 -- -- -- -- -- -- 45-60 4.9 43.4 30.6 0.357 0.052 -- -- -- -- -- -- 60-75 nd nd nd 0.193 0.037 -- -- -- -- -- -- 75-90 4.6 55.7 23.9 0.15 0.034 -- -- -- -- -- -- Prince 90-105 nd nd nd 0.151 0.033 -- -- -- -- -- -- Georg es I 105-120 4.5 69.6 20 0.187 0.031 -- -- -- -- -- -- 120-135 nd nd nd 0.353 0.044 -- -- -- -- -- -- 135-150 4.5 69.6 20.1 0.12 0.028 -- -- -- -- -- -- 150-165 nd nd nd 0.103 0.025 -- -- -- -- -- -- 165-180 4.4 78.3 14.9 0.103 0.023 -- -- -- -- -- -- 180-195 nd nd nd 0.091 0.020 -- -- -- -- -- -- 195-210 4.4 77.6 13.6 0.104 0.022 -- -- -- -- -- -- 1 pH by glass combination pH electrode and a pH meter (Metler Toledo InLab®413 combination meter). 2 Soil particle size analysis by the Modified Pipette Method. Gavlak, R., D. Horneck and R.O. Miller. 2005. Particle size analysis modified pipette method. Soil, plant and water reference methods for the western region. 3rd ed. 3 Total C and N analysis at University of Maryland Department of Environmental Science and Technology Analytical Lab on LECO CN628 Elemental Analyzer (LECO Corp., St. Joseph, MI; Nelson and Sommers, 1996; Matejovic, 1993). 4 Soil organic matter (SOM) (Loss On Ignition method) and nutrient content by Mehlich3 extraction (P, K, Mg, Ca, Na, S) at WayPoint Analytical, Inc (Richmond, VA). 196 Appendix 8. Cover crop sampling details Table 28. Fall and spring cover biomass number and size of quadrats collected from each plot. Farm Fall quadrats Spring quadrats Franklin I 2 x (0.25 m2) 2 x (0.25 m2) Franklin IIA 2 x (0.25 m2) NA Frederick I 2 x (0.25 m2) 2 x (0.25 m2) Frederick III -- 2 x (0.25 m2) Harford I 2 x (0.25 m2) -- Howard IA 3 x (0.25 m2) 3 x (0.25 m2) Huntington IA 2 x (0.25 m2) 2 x (0.25 m2) Lancaster IA 2 x (0.25 m2) 2 x (0.25 m2) Lancaster II 2 x (0.25 m2) 2 x (0.25 m2) Lancaster III -- 2 x (0.25 m2) Prince Georges I 2 x (0.25 m2) 2 x (0.25 m2) Franklin IIB 3 x (0.25 m2) -- Frederick IV 5 x (0.25 m2) 3 x (0.25 m2) Howard IB 3 x (0.25 m2) 3 x (0.25 m2) Huntington IB 2 x (0.50 m2) -- Kent II 4 x (0.25 m2) 3 x (0.25 m2) Dorchester IB 4 x (0.25 m2) 3 x (0.25 m2) Lancaster IB 3 x (0.25 m2) 3 x (0.25 m2) Lancaster V 5 x (0.25 m2) 3 x (0.25 m2) 197 Appendix 9. List of weather stations Table 29. Distance from weather station to farm for precipitation and temperature measurements. Distance to weather station Farm Precipitation Temperature km Caroline I 5.86 35.7 Dorchester IA 8.01 11.9 Franklin I 4.80 4.80 Franklin IIA 4.85 4.85 Frederick I 5.83 18.6 Frederick III 0.89 22.9 Harford I 13.7 13.7 Howard IA 5.20 9.78 Huntington IA 7.09 27.7 Kent I 17.0 30.4 Lancaster IA 7.99 7.99 Lancaster II 2.20 9.31 Lancaster III 7.03 28.0 Prince Georges I 4.43 4.43 Dorchester IB 1.61 12.1 Franklin IIB 4.64 4.64 Frederick IV 3.69 21.9 Howard IB 6.03 9.27 Kent II 11.3 19.1 Lancaster IB 10.1 10.1 Lancaster V 5.06 5.06 198 Appendix 10. Bulk density of soil cores The number of cores that were averaged and the depth increments varied by farm (Table 30). The bulk density values for each layer from each farm were analyzed using box and whisker plots in SAS version 9.4 (SAS Institute, Cary, NC). To exclude outliers and possible errors, values beyond 1.5 x the interquartile range (25th to 75th percentiles) were not included when calculating the average bulk density value. Table 30. Bulk density (BD) mean (g cm-3), standard deviation (SD) (g cm-3), first (Q1) and third (Q3) interquartile range (25th to 75th percentiles) and number of outliers above fences for all farms in which soil cores were taken. The fence is defined as 1.5 x Interquartile range (25th to 75th percentiles). # of outliers Soil depth, # of cores BD mean, BD SD, g above Site cm averaged g cm-3 cm-3 BD Q1 BD Q3 fences Dorchester IB 0-60 36 1.502 0.125 1.43 1.59 1 60-120 36 1.72 0.213 1.60 1.87 1 120-180 35 1.53 0.297 1.37 1.81 1 Frederick I 0-120 29 1.63 0.272 1.54 1.66 1 120-210 25 1.87 0.227 1.71 2.02 1 Frederick III 0-120 20 1.22 0.187 1.12 1.37 1 120-210 19 1.48 0.359 1.24 1.74 1 Frederick IV 0-60 24 1.39 0.0780 1.33 1.47 1 60-120 23 1.54 0.138 1.46 1.63 1 120-210 23 1.38 0.275 1.28 1.62 1 Harford I 0-120 15 1.31 0.273 1.16 1.49 1 120-210 15 1.19 0.362 1.02 1.44 1 Howard IA 0-120 9 1.43 0.0881 1.35 1.49 1 120-210 6 0.985 0.281 0.782 1.28 1 Howard IB 0-60 25 1.31 0.0634 1.26 1.34 1 60-120 25 1.48 0.110 1.38 1.57 1 120-210 27 1.51 0.280 1.38 1.64 1 Huntington IA 0-120 32 1.27 0.385 0.864 1.59 1 120-210 30 1.39 0.427 1.02 1.69 1 Kent II 0-60 24 1.53 0.124 1.44 1.62 1 60-120 24 1.62 0.0936 1.58 1.66 1 120-210 23 2.08 0.512 1.56 2.65 1 Lancaster IA 0-120 29 1.22 0.217 1.08 1.43 1 120-210 28 1.48 0.240 1.35 1.67 1 Lancaster IB 0-60 26 1.14 0.180 1.01 1.23 1 60-120 25 1.39 0.175 1.28 1.49 1 120-210 26 1.44 0.312 1.25 1.57 1 199 Lancaster II 0-120 32 1.05 0.158 1.01 1.15 1 120-210 32 1.12 0.179 1.06 1.26 1 Lancaster III 0-120 20 1.36 0.263 1.23 1.54 1 120-210 18 1.57 0.341 1.34 1.75 1 Lancaster V 0-60 40 1.30 0.0814 1.25 1.36 1 60-120 40 1.63 0.115 1.57 1.70 1 120-210 38 1.50 0.323 1.35 1.70 1 200 Appendix 11. Corn yield equations Equation 19 Corn silage yield 𝑤ℎ𝑜𝑙𝑒 𝑝𝑙𝑎𝑛𝑡 𝑑𝑟𝑦 𝑤𝑡 𝑆𝑖𝑙𝑎𝑔𝑒 𝑦𝑖𝑒𝑙𝑑 = ℎ𝑎𝑟𝑣𝑒𝑠𝑡 𝑎𝑟𝑒𝑎 Where, (100−𝑤ℎ𝑜𝑙𝑒 𝑝𝑙𝑎𝑛𝑡 % 𝑚𝑜𝑖𝑠𝑡.) 𝑤ℎ𝑜𝑙𝑒 𝑝𝑙𝑎𝑛𝑡 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝑤ℎ𝑜𝑙𝑒 𝑝𝑙𝑎𝑛𝑡 𝑤𝑒𝑡 𝑤𝑡 ∗ 100 𝑠𝑢𝑏𝑠𝑎𝑚𝑝𝑙𝑒 𝑑𝑟𝑦 𝑝𝑙𝑎𝑛𝑡+𝑒𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑤ℎ𝑜𝑙𝑒 𝑝𝑙𝑎𝑛𝑡 % 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 = (1 − ) ∗ 100 𝑠𝑢𝑏𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑡 𝑝𝑙𝑎𝑛𝑡+𝑒𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 Harvest area = row width x length of harvest area Equation 20 Corn grain yield 𝑔𝑟𝑎𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 15.5% 𝑚𝑜𝑖𝑠𝑡. 𝐺𝑟𝑎𝑖𝑛 𝑦𝑖𝑒𝑙𝑑 = ℎ𝑎𝑟𝑣𝑒𝑠𝑡 𝑎𝑟𝑒𝑎 Where, 𝑠𝑢𝑏𝑠𝑎𝑚𝑝𝑙𝑒 𝑑𝑟𝑦 𝑔𝑟𝑎𝑖𝑛 𝑤𝑡 15.5 𝑔𝑟𝑎𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 15.5% 𝑚𝑜𝑖𝑠𝑡. = 𝑤𝑒𝑡 𝑒𝑎𝑟 𝑤𝑡 ∗ ∗ 𝑠𝑢𝑏𝑠𝑎𝑚𝑝𝑙𝑒 𝑑𝑟𝑦 𝑒𝑎𝑟 𝑤𝑡 𝑒𝑎𝑟 % 𝑚𝑜𝑖𝑠𝑡. 𝑑𝑟𝑦 𝑒𝑎𝑟 𝑤𝑡 𝑒𝑎𝑟 % 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 = (1 − ) ∗ 100 𝑤𝑒𝑡 𝑒𝑎𝑟 𝑤𝑡 201 Chapter 5: Policy implications and Conclusion Policy Implications—improving efficiency of cover crop program through deep rooted cover crops Currently the State of Maryland has an incentive program in which landowners are paid to grow cover crops. The incentive payment amounts vary, depending on cover crop species, cover crop planting date, previous cash crop, and field management practices. Farmers are eligible for cover crop payments if they plant the cover crop by 5 Nov and kill after 28 Feb. The program gives “early planting” bonus payments if the cover crop is planted before 15-October (Maryland Department of Agriculture, 2018). We found in the current study that cover crops planted after 30-September will have minimal biomass accumulation and soil N uptake and will not capture subsoil N in the fall. Under the current cover crop program, planting rye alone is given a bonus incentive over planting rye within a mix. However, we found in the current study that rye monocultures typically require additional spring N fertilization or decrease subsequent corn yields. If planting cover crops leads to increased N fertilizer requirements, it is counterproductive toward the goal of using cover crops to reduce residual soil N and risks of N leaching from cropland. The EPA Interim Evaluation of Maryland’s 2016-2017 milestones reports that the Agriculture sector in Maryland was not on-track to reach its 2017 N target, which is a 60% reduction of the 2009 N loads into the Bay to achieve water quality standards (EPA, 2017). While this failure may be partly a result of legacy N effects due to the slow flow of groundwater (Ator and Denver, 2015), it may also be partly a result of conservation 202 practice implementation. For example, cover crops will not perform to their full potential if they are planted too late. Due to the extent of cover crops on the landscape (e.g., 478,000 acres in Maryland in 2014), improvements in the ability of cover crops to reduce N leaching from the land through incorporating earlier planting dates and more deep- rooted species could foster a more sustainable, cycling crop system and greatly reduce the N load into bodies of water. We therefore suggest that incentives be increased for earlier cover crop planting, especially for planting prior to mid-September. We recognize that such early cover crop planting may require additional adaptations of a farm system, such as earlier maturing crop varieties, interseeding into standing crops, or changes in crop rotations. Overall conclusions We found there were often trade-offs between scavenging residual N and releasing the N for the subsequent crop. Radish was very effective at scavenging N in the fall; however, it sometimes led to increased levels of NO3-N in the spring soil in shallow as well as deep soil layers (e.g., Lancaster III and Lancaster V farms in Figure 16). The 15N tracer study indicated that radish, rye, and two-way or three-way mixes of radish + rye + (crimson clover) all performed equally well in scavenging residual N from deep soil layers. Winter cereal cover crops caused a yield loss and/or increased N fertilization needs for a subsequent corn crop. Utilizing mixed cover crop species may be optimal in terms of N scavenging and release. Mixed cover crops were very effective at scavenging residual N in the fall and spring, and typically did not cause a yield gain or loss for subsequent crops. Radish may 203 also be a good choice, in terms of being able to reduce subsequent corn N fertilizer application amounts. The effect of cover crops on the cropping system may take more than one growing season to become apparent. For example, the phenomenon observed on Lancaster IA, where there was no corn yield response to N fertilizer, may have resulted from the long-term cumulative effects of many years of mixed-species cover cropping and manure applications on this farm. Furthermore, it is important to keep in mind that cover crop effects are expected to be highly variable from site-to-site and year-to-year, according to soil and weather patterns. Thus, while the on-farm proponent of our study was not as precisely controlled as the research station trials, we believe it was very valuable for considering the range of cover crop responses that we would expect to see across practitioners. In conclusion, we found substantial levels of inorganic soil N remained in the soil profile (0-210 cm deep) following summer crops. On average, there was more residual inorganic N in the soil profile than the amount of N fertilizer that a farmer would typically apply to a corn crop. This provides both a risk and an opportunity. The residual N is at risk to leach into bodies of water and cause eutrophication and associated environmental problems. However, the pool of residual N also could serve as a valuable resource to farmers, if they utilize it and reduce their fertilizer use. Cover crops were able to access deep pools of N, but only if the cover crops were planted by the first week in September in our study region. 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