The study was conducted at Mississippi State Plant Science Center, on a Leeper silty clay loam (fine, smectitic, nonacid, thermic Vertic Epiaquepts) soil in the fall of 2019 and had been continually conducted on the same plots for 4 years (2019-2022). Mean monthly temperature, total monthly and 30-year normal rainfall data were obtained from a weather station at the Plant Science Center, Mississippi State University, as shown in Table 1. A randomized complete block split-plot design with three replicates was used in this study. The four main plots treatments included winter fallow (WF) (no cover crop), inoculated crimson clover (CC) (Trifoliumincarnatum), Elbon cereal rye (CR) (Secale cereale L), and cereal rye + crimson clover (Mix). No fertilizer, inorganic fertilizer and fall-applied broiler litter (BL) were the sub-plot treatments. Each subplot was 4 rows wide by 9 m length with a row spacing of 1 m. Each year, broiler litter at the rate of 4.5 Mg/ha was applied surface broadcast after cover crop establishment and to WF using a calibrated manure spreader. There were approximately 32 g total N/kg, 18 g total P/kg, and 28 g total K/kg in BL on average. Cereal rye (CR) and CC were planted with a John Deere 1560 drill (Deere & Company, Moline, Illinois, USA) with a row spacing 20 cm parallel to crop rows. Crimson clover and CR were seeded at a rate of 35 and 120 kg ha⁻¹, respectively. The rates for CR and CC in the Mix were 60 and 18 kg/ha, respectively. The seeding rate was selected to optimize biomass production and suppress weeds. Cover crops were planted approximately mid-September through mid-October. In the spring, the cover crops were terminated by spraying 1.5 kg/ha of the active ingredient glyphosate (N-(phosphonomethylglycine). After termination, in 2020 and 2021 corn cultivar Dekalb 63-84 and in 2022 Dekalb 66-40 was planted using precision No-Till planter (John Deere, Moline, Illinois, USA). With WF and cover crops, plots received BL, supplemented with inorganic fertilizer at the rate of 80 kg N/ha at V6 corn growth stage. With inorganic fertilizer plots, corn received 224 kg N/ha as UAN (urea ammonium nitrate, 32% N), applied as side-dress split application: 56 kg N/ha at planting and 168 kg N/ha at the V6 growth stage, as recommended by the Mississippi Soil Testing Laboratory. Fertilizer was injected 5 cm to the side and 10 cm deep using a liquid applicator.

Table 1. Total monthly and 30-year normal rainfall (mm) received and temperature (˚C) at the Plant Science Center, Mississippi State, MS from 2019 to 2022.

Before initiating the experiment and before planting cover crops in the fall of 2019, composite soil samples were collected using a 2.5-cm diameter probe at depths of 0 - 15 cm. Soil samples were air-dried and analyzed for soil chemical and physical properties. Soil pH was determined by using a pH meter with a 1:1 mixture of soil and 0.05 m calcium chloride solution (Fisher Scientific, Pittsburgh, PA, USA) (Reference). Total C and N content were analyzed using an elemental CN analyzer, the Vario Max Cube (manufactured by Elementar American Inc., Mt. Laurel, NJ, USA). Soil cations and anions were extracted using 1:10 ratio (2.5 g soil :25 ml M3 solution), shaken for 5 min (Mehlic 3, 1984), filtered and analyzed by inductively coupled argon plasma spectrophotometer (ICP 9000) (Thermo Jerrell Ash, Franklin, MA).

Soil bulk density was measured using a hammer-driven core sampler with an inner ring diameter of 5.7 cm at 0 - 15 cm depth. The weight of each field-moist sample was recorded. Soil was oven-dried at 105˚C for 48 hours. Soi bulk density was calculated by dividing the dry weight by the volume of the soil, as described by Blake and Hartge (1986) [25]. Soil cation exchange capacity (CEC), number of exchangeable sites within a soil for cations, was determined using ammonium acetate, buffered at pH = 7, compulsory displacement method [26]. Initial soil nutrient concentrations were, total C and total N were 11.6 g/kg and 0.83 g/kg, respectively and extractable soil phosphorus (P) and potassium (K) were 52 and 184 mg/kg, respectively. The soil bulk density was 1.27 g/cm³ and soil CEC was 42 cmol_c/kg.

Broiler litter sample was taken before application by collecting multiple samples from different locations within the BL delivered. These samples were then combined and mixed to obtain a representative sample for determining nutrient content and ensuring optimal nutrient delivery to corn. Broiler litter samples were dried, ground to pass a 2 mm sieve and analyzed for total N and C content using an elemental CNS analyzer (described above) with dry combustion. Total P, K, Ca, and Mg contents of BL were assessed by dry-ashing a 1.0-g sample, following the methods outlined by Isaac and Kerber (1977) [27]. These elements were then quantified using an ICP. The chemical characteristics of BL are summarized in Table 2.

Table 2. Broiler litter chemical characteristics and total amounts nutrients applied with application of broiler litter at the rate of 4.5 Mg ha⁻¹y⁻¹.

In April of each year, prior to termination of cover crops, four randomly selected 0.5 m square quadrat samples of cover crop aboveground biomass were harvested from each cover crop plot. Samples were cut with pruning shears approximately 1.0 cm above the soil surface, dried at 65˚C and dry weight was recorded. Dried samples were ground through a Wiley mill to pass a 1-mm screen. Ground samples were re-dried for another 24 h and sealed in vials. Samples were weighed and analyzed for total C and N analysis using an elemental CN analyzer (described above). The total N content in the cover crop aboveground biomass was calculated by multiplying the dry matter collected in the sampled squares by the corresponding N concentration. After the cover crop was fully desiccated, soil cores were collected from each plot using a 2.5-cm diameter soil probe at a depth of 0 - 15 cm just before planting corn. Soil samples were air dried, ground, and extracted with 2M KCL and analyzed for NO₃ and NH₄ using a Lachat instrument (QC 8000 flow injection analyzer; Lachat, Loveland, CO). At R1corn growth stage (silking), 3 soil samples were collected at 0 - 15 cm depth, thoroughly mixed, one composite sample was taken and analyzed for NO₃-N concentration. The field operation information is shown in Table 3.

Table 3. Field operations in a four-year corn study at Plant Science Center, Mississippi State, MS.

UAN: urea ammonium nitrate.

At physiological maturity, whole corn plant was collected from 0.3 m² in one of the border rows in each plot, oven dried at 65˚C and dry matter was recorded. Corn grain was harvested from the center two rows the whole length of each plot using an automated plot combine. All reported grain yield was adjusted to 15.5% moisture content. At harvest, a sample of grain, approximately 400 g, was collected from each plot, dried, ground, and analyzed for grain N concentration using CN dry combustion analyzer. Grain P, K, Cu and Zn contents were determined by dry-ashing a 1.0-g sample, following the methods outlined by Isaac and Kerber (1977). These elements were then quantified using an ICP. Additionally, the total grain nutrient uptake was calculated as the product of the nutrient concentration and the total grain yields. After corn harvest, on 15 September in each plot, 3 soil samples were collected at 0 - 15, 15 - 30- and 30 - 60 cm depth, thoroughly mixed by depth, and one composite sample was taken for each depth per plot, air-dried, ground, and analyzed for residual NO₃-N.

Data was analyzed using a MIXED model procedure with block and block by cover crop as random effects and cover, fertilizer, and cover by fertilizer interaction as fixed effects in Statistical Analysis System (SAS) [28]. The mixed-model procedure provides Type III F values for cover using block by cover as error and fertilizer and fertilizer by cover using as residual error. Corn grain yield, grain nutrient concentrations, pre-planting, and post-harvest soil NO₃-N were considered dependent variables, while cover cropping and fertilization were considered independent variables in the analysis. Year was treated as a fixed effect to determine interactions involving year. If cover crop by fertilizer interaction is not significant, the main effects are reported but if the interaction is significant, the simple effect of fertilizer within each cover level or viseversa are reported. Mean separation was evaluated through a series of pairwise contrasts among all treatments. Main effects and all interactions were considered significant when P ≤ 0.05.

Rainfall quantity and distribution varied across years (Table 1). Cumulative rainfall during the corn growing season (April through August) was 644 mm in 2020, 892 mm in 2021, and 515 mm in 2022. Over the three-year period, rainfall fluctuated, with 2022 and 2020 receiving approximately 28% and 42% less rainfall than 2021, respectively. The total rainfall received during the critical corn growth stages (flowering to grain filling) in June and July was 241 mm in 2020, 451 mm in 2021, and 136 mm in 2022. Mean air temperatures in June, July, and August were 27.0˚C, 26.6˚C, and 27.8˚C in 2020, 2021, and 2022, respectively (Table 1). From June to August, the mean monthly temperatures in 2020 and 2022 were comparable to the historical average of 27.9˚C. These months were generally warmer and drier in 2020 and 2022, suggesting that plants were exposed to relatively hot and dry conditions, particularly during flowering and grain filling.

Dry matter residues produced by winter cover crops play an important role in preserving soil moisture and suppressing weeds [29] during the corn growing season. Cover crop DM, C and N concentrations, and C/N ratio were significantly affected by year (Table 4). Cumulative rainfall throughout the cover crop growing season (October to April) was 1189 mm in 2019/2020, 775 mm in 2020/2021, and 558 mm in 2021/2022. Averaged across cover crop types and fertilization treatments, the magnitude of winter cover crop DM, was greater during the first growing season (2019/2020) (9.4 Mg/ha) than during the second (2020/2021) (6.5 Mg/ha) and third (2021/2022) (5.8 Mg/ha) growing seasons. Due to the significant interaction between winter cover crops and year (Table 4), treatment effects on DM, N and C contents, and the C/N ratio are presented separately by year, meaning rerun the model with year taken out to determine the means and mean separation. Averaged across fertilization, CR produced greater amounts of DM than those produced by CC and the cover crop mixture in 2020, but no differences in DM yield was obtained between CR and the mixture in 2021 and 2022 (Table 5). Crimson clover DM yield was smaller than those produced by CR and the mixture in 2021 and 2022 (Table 5). The greater biomass produced by CR is most likely related to better growth overwinters, which enables it to form a physical barrier that suppresses weeds by producing large amounts of biomass [30]. The lower DM yield in CC in 2021 and 2022 may be related to lower temperatures in December and January of 2020/2021 (7.4˚C and 5.8˚C) and 2021/2022 (7.2˚C and 6.1˚C) (Table 1), which likely led to stand reduction and decreased biomass production. In agreement with our results, Thurston et al. (2022) [31] reported that cold stress, especially when temperatures drop below 10˚C, reduces nodulation and symbiotic nitrogen fixation (SNF) in winter annual legumes. In contrast to DM yield, N content in CR was significantly less than CC (Table 5). This contrast between nitrogen (N) content and DM production in CR versus CC is a classic example of how plant physiological roles shape cover crop performance. For example, CR scavenges possible residual soil nitrogen left over from previous crops limits its N content to what’s available in the soil [30]. In contrast, CC fixing atmospheric nitrogen allows it to accumulate higher N concentrations in its tissues. However, in 2022, N content by above ground biomass was greater with the mixed cover crop than individual CR and CC species (Table 5). This may have resulted from the N supplied by CC, thereby increasing N concentration in the mixture, reduced the CN ratio as compared to CR alone (from 35 to 16) (Table 5). In agreement with the results, Ranells and Wagger (1996) [19] reported when cereal rye was grown in bicultural with crimson clover, increased N content by the biomass and the C:N ratio reduced from 40:1 in cereal rye grown in monoculture to 28:1 when cereal rye grown with crimson clover has less likelihood of immobilizing N during decomposition. Averaged across cover crop species, the main effect of BL significantly increased aboveground biomass in 2020 and 2021 compared to the control and inorganic fertilizer (Table 5). Since inorganic fertilizer was not applied to established cover crops in the fall, no differences in DM yield were observed between inorganic fertilizer and the control in 2020 (Table 5). The means of DM yield added to the soil system across all three years was significantly greater with cereal rye (8.7 Mg/ha) than with crimson clover (6.5 Mg/ha) or combined cereal rye and crimson clover (6.6 Mg/ha). The contribution of cover crop residues can potentially enhance soil structure by creating and modifying macropores, leading to better soil aggregation [32] and microbial activity [33], thereby improving root growth and overall plant vigor, which leads to greater total biomass (Table 5). Averaged across fertilization treatments, the main effects of cover crops on C and N concentrations and contents were evaluated. Carbon concentration was significantly greater in CR compared to CC and mixed cover crops across all years (Table 5), following the order of CR > the mixture > CC. The greater C content in CR plots than in CC plots resulted from the greater biomass yield of CR, as C content is a product of cover crop biomass and C concentration. Conversely, the greater N content in CC plots compared to CR plots was primarily due to the greater N concentration in CC. In the first two years, the mixed cover crop had lower C content than CR but comparable C content to CC. However, in 2022, the cover crop mixture yielded higher dry matter, C, and N contents than either species grown alone (Table 5). This was due mainly to the effect of legumes on CC, which could fix biological nitrogen, improve nutrient cycling, and promote nitrogen transfer to CR [34], thereby increasing the biomass yield and N concentration of CR in the mixed cover crop, as also reported by Ranells and Wagger (1996). Although non-legume cover crops, such as CR, scavenge nitrogen (N) and reduce N leaching [35], they typically have lower N concentrations and higher C/N ratios compared with legume cover crops, thereby providing less N required for crop production [36]. The C/N ratio of CR ranged from 33 to 39 in 2020 and 2021, while the C/N ratio of CC ranged from 11 to 12 (Table 5). When the CC was grown with CR in the cover crop mixture, the C/N ratio dropped to a range of 13 to 14 in 2020 and 2021. As a result, the potential of CR in the mixture, compared with CR alone, to immobilize soil N was reduced, probably due to the differing proportions of CC and CR in the mixture. This likely resulted in the release of more N from the cover crop mixture residue in the soil than from CR residue alone, as evidenced by greater N content in the mixed cover crop in 2022 (Table 5). Therefore, a legume-cereal combination can reduce the C/N ratio of CR cover crops and increase the potential for soil N mineralization and availability for the succeeding crop. Averaged across cover crop treatments, the main effect of fertilization treatments did not have any significant effects on cover crop C concentrations in 2020 and 2021. However, in 2022, the main effect of BL significantly increased cover crop C concentration compared to the unfertilized control treatment. Nitrogen concentration showed different patterns, being greater in CC than in CR and the mixed cover crops in 2020 and 2021, and similar between the mixed and CC cover crop alone in 2022.

As a result, the C/N ratio was higher in CR and lower in CC and the mixed cover crop (Table 5). The effects of cover crops and BL treatments on total N content of residues were like the dry matter results, with cover crop responses varying by year. The effect of poultry litter was significant, while no interactions were observed between cover crops and fertilization treatments. Overall, CC and the mixed cover crop resulted in the greatest N contents in all three years (Table 5).

Averaged across cover crop species, fertilization followed a similar pattern to cover crop species, where the main effect of broiler litter had a greater impact on cover crop N concentration than the main effects of inorganic fertilizer and unfertilized control in 2020 and 2021 (Table 5). In 2022, no differences in cover crop N concentration were observed between the main effects of broiler litter and inorganic N fertilizer, but both resulted in greater cover crop N concentration than the unfertilized control. As with biomass yield, averaged across fertilization treatments, the C content in CR and in the mixed cover crop were similar in 2021 and 2022, however, N content in the mixed cover crop was greater and C/N ratio was lower than that in CR cover crop in 2022 (Table 5).

Table 4. Probability levels from analysis of variance (ANOVA) associated with the treatments’ effects on cover crop dry matter yield, N and carbon concentrations and uptake and C/N ratio, Plant Science Center, Mississippi State, Ms.

Table 5.Effects of fall-applied broiler litter on cover crop dry matter production and carbon and nitrogen uptake at the Plant Science Center, Mississippi State, MS.

Different letters in a column indicate a significant difference between soil amendments or cover cropping system at P≤ 0.05 level.

Nitrate-N concentrations in the soil were measured from samples collected at a depth of 0 - 15 cm throughout the experiment. The NO₃-N results are presented in two phases: Phase 1, occurring prior to corn planting or approximately 30 days after cover crop termination; and Phase 2, corresponding to the R1 growth stage, which marks the onset of silking and kernel formation (Figure 1).

Figure 1. Soil NO₃-N in 0 - 15 cm depth at corn planting (A) and at R1 corn growth stage (B) as affected by cover crop species and fertilization treatments averaged across years (2020-2022).

Pre-plant soil NO₃-N content in the top 15 cm (phase 1) was significantly influenced by cover crop type, fertilizer source, and their interaction (Table 6). Because the interaction between winter cover crops × year (P = 0.1677) and year × fertilization (P = 0.8567) were not significant, soil NO₃-N data collected each year at corn planting and during the R1 growth stage were averaged across years. The resulting mean values are presented in Figure 1. The vertical lines between fertilizer treatments and cover crops indicate comparisons of the main effects of these treatments on soil NO₃-N concentrations.

Averaged across fertilization treatments, soil NO₃-N levels at the corn planting stage were greater in CC plots than in CR plots, likely due to differences in N cycling between the two cover crops (15.8 mg/kg vs. 7.9 mg/kg (Figure 1). The greater NO₃-N levels in CC plots were most likely related to the rapid decomposition of residues after termination, driven by the low carbon-to-nitrogen (C/N) ratio of 12. This accelerated breakdown released nitrogen into the soil, enhancing early-season N availability, as also reported by Büchi et al. (2015). In contrast, CR residues, with a high C/N ratio of 36, decomposed more slowly. This slower breakdown initially immobilized nitrogen as soil microbes processed the residues, temporarily reducing N availability at planting (Figure 1).

Although higher NO₃-N levels were recorded in CC and mixed cover crop treatments compared to the controls, no significant differences in NO₃-N concentrations were observed among the control, CC, and the mixed cover crop at corn planting (P > 0.05). In contrast, pre-planting soil NO₃-N concentrations were significantly lower in CR plots (Figure 1). According to Lacey et al. (2022) [37] and Roth et al. (2022) [38], only 10% of the nitrogen scavenged by CR cover crops and assimilated in the aboveground residue was subsequently recovered in the corn crop. The reduced nitrogen release from CR residues at planting is likely due to nitrogen immobilization, driven by its high carbon-to-nitrogen (C/N) ratio (Table 5).

The findings are consistent with those of by Ranells and Wagger (1997), who reported that cover crop residue can influence N availability to subsequent crops, with high C/N ratios posing a risk of short-term N immobilization and potential due to high C/N ratios posing a risk of short-term and potential yield reduction. Soil NO₃-N levels with CC cover crop plots were 36% and 60% greater than those observed in no cover crop and CR treatments, respectively (Figure 1). The lower NO₃-N levels in no winter cover crop were likely due to nitrogen losses, through leaching or denitrification in winter fallow.

At corn planting, there was no significant difference in soil NO₃-N between the cover crop mixture (13.7 mg/kg) and crimson clover (15.4 mg/kg) (Figure 1). Averaged across cover crop treatments, the mean pre-planting soil NO₃-N content with broiler litter application was 29% and 45% greater than with inorganic N fertilizer (15.4 vs. 11.6 mg/kg) and the unfertilized control (15.4 vs. 8.7 mg/kg), respectively (Figure 1). Although inorganic N fertilizer was not applied to established cover crops in the fall, the greater pre-planting soil NO₃-N in inorganic N fertilizer plots compared to unfertilized plots (11.6 vs. 8.7 mg/kg) may be attributed to residual soil N left after the previous corn harvest.

At the R1 corn growth stage, N tied up in microbial biomass as organic N appears to have started becoming available through mineralization. At R1, NO₃-N levels were significantly (P < 0.05) higher in all cover crop treatments compared to controls; however, the cover crop treatments were not significantly different from one another (Figure 1). This is likely related to supplemental inorganic N fertilizer applied to all cover crop treatments during the V6 corn growth stage, at a rate of 80 kg N/ha, to prevent potential N deficiencies.

Although the differences were not significant, soil NO₃-N levels were generally greater with CR and Mixed cover crops compared to CC cover crops (Figure 1). The lower NO₃-N concentrations in CC cover crops at the R1 corn growth stage were likely due to the rapid decomposition rate of residues, which released N into the soil before corn planting. In contrast, a substantial portion of CR and mixed cover crops residues remained on the soil surface throughout the corn growing season. Thus, N released from CC residues may have leached beyond the root zone, or lost through denitrification, leading to reduced NO₃-N levels relative to CR and mixed cover crops.

These findings align with Nagumo and Nakamura (2013) [39] who observed that only 5% of hairy vetch residue remained at the end of corn season. Similarly, Lee et al. (2002) reported that legumes, particularly vetch, lost 72% - 81% of their initial weight within one month after termination, concluding that crimson clover could serve as a nitrogen starter fertilizer but not a sustainable N source due to its rapid decomposition. The results with CC cover crop highlight the rapid mineralization and early release of mineral N due to its C/N ratio of 12 (Table 5). However, CR cover crop residue decomposed more gradually, with a sustained release of mineral N due to its high C/N ratio of 36 (Table 5), better synchronizing with the development of corn plants.

Ruffo and Bollero (2003) found that CR cover crops were a more suitable material than vetch for N supply and soil conservation. The addition of CR cover crops resulted in more sustained release of mineral N, and with timing that better matched crop N requirements during early growth stages. The slower N release from CR cover crops make it a more effective cover crop for reducing NO₃-N leaching and mitigating water pollution [40]. Therefore, from a soil fertility perspective, CR cover crops can be considered as a better cover crop option than legumes such as CC but need to be fertilized to minimize N deficiency and prevent corn yield reduction. However, mixed cover crop did not differ significantly from CR cover crop alone.

At the R1 corn growth stage, CR cover crop and the cover crop mixture exhibited the greatest levels of soil NO₃-N (11.8 mg/kg), as compared to CC cover crops alone (Figure 1).These results are consistent with findings by De Notaris et al. (2018), who reported that mixtures of legumes and non-legumes reduced nitrate leaching by approximately 60% in loamy sand soils under temperate conditions. Averaged across cover crops, soil NO₃-N at the R1 corn growth stage was greater with the application of broiler litter (11.4 mg/kg) compared to inorganic N fertilizer (9.6 mg/kg and the unfertilized control (8.7 mg/kg) (Figure 1).

Table 6. Probability levels from analysis of variance (ANOVA) associated with the treatment’s effects on corn grain yield, grain nutrient uptakes, Plant Science Center, Mississippi State, MS.

In the comprehensive analysis, interaction between year × fertilization and year × cover crop was statistically significant for both grain yield and grain N uptake (Table 6). Therefore, the effects of treatments on corn grain yield and grain N uptake were presented and discussed separately for each year. Averaged across cover crops and fertilization treatments, corn grain yield in 2022 was 58% lower than in 2020 (3634 vs. 825 kg ha⁻¹) and 45% lower than in 2021(3634 vs. 8027 kg ha⁻¹) (Table 7).

This reduction in grain yield in 2022 likely reflected poor growing conditions, primarily due to water stress during June and July—critical growth stages for corn, including tasseling, silking, pollination, and kernel development. Any level of water stress during this phase can significantly reduce grain yield. Total rainfall during June and July was 136 mm in 2022, compared to 241 mm in 2020 and 451 mm in 2021 (Table 1). The markedly lower rainfall in 2022 contributed to poor pollination, kernel abortion, and reduced grain weight, ultimately resulting in diminished yields, as previously reported by Shaw (1988) [41].

Averaged across fertilization treatments, the main effect of cover crops did not significantly influence corn grain yield in 2020 and 2021. However, in 2022, the main effect of cover crops significantly influenced corn grain yield (Table 7), suggesting that corn responded to cover crop residues under drier conditions. These residues are likely to help conserve soil moisture, mitigating the impact of water stress during the growing season. The results are consistent with Alvarez et al. (2017) [42], who found that maize yield response was higher in areas with <500 mm annual rainfall compared to areas with rainfall ranging from 500 to 700 mm during the growing season. Thus, low soil moisture conditions can improve the growth and increase associated benefits of cover crops with poultry litter by reducing nutrient leaching, which in turn can positively affect early-season growth, development, and yield of succeeding main crops [43].These findings are consistent with those of Tewolde et al. (2015) [44], who observed minimal effects of cover crops on cotton lint yield during wetter years, but reported enhanced lint yield under suboptimal rainfall conditions.

In 2022, averaged across fertilization treatments, the main effects of CR cover crop and the mixed cover crop significantly increased corn grain yield as compared to CC cover crop (Table 7). The greater corn grain yield in CR and mixed cover crop treatments than CC or no cover crop most likely related to N uptake. Nitrogen uptake was significantly greater with mixed cover crop than in the other cover crop treatments (Table 7). These findings suggest that less N input may be sufficient for corn grown in legume/non-legume cover crop mixtures. The effectiveness of cover crops in reducing residual soil nitrogen accumulation depends on their ability to establish quickly in the fall, the extent of their root systems, and their biochemical composition—particularly the carbon-to-nitrogen ratio [45].

The lower grain yield associated with CC cover crops was likely due to limited nitrogen mobility, which was hindered by insufficient soil moisture during June and July (Table 1). Because approximately 90% of legume biomass decomposes within one month of termination due to rapid breakdown, the absence of CC residue on the surface during these critical months failed to conserve soil moisture through evaporation suppression. As a result, corn plants were unable to effectively utilize soil-available nutrients, leading to reduced grain N uptake and lower yields (Table 7).

These findings are consistent with previous studies reporting that cover cropping can negatively impact main crop yields by reducing the availability of inorganic nitrogen for crop uptake (Kaspar & Bakker, 2015) [46]. However, at the same Mississippi State location with similar soils, Seman-Varner et al. (2017) [47] reported that corn grain yield was greatest under legume cover crops, followed by legume-rye mixtures, and lowest under cereal rye, highlighting the context-dependent nature of cover crop effects on yield outcomes. This may be because, weather-driven changes in clover biomass, N release, soil moisture, and microbial activity can easily cause corn to respond positively in one year and negatively in another.

Averaged across years, the main effects of CR and the mixed cover crops increased corn grain yield by 20% as compared to crimson clover (Table 7). No significant differences in corn grain yield were observed between the main effects of CR and mixed cover crop (Table 7). In contrast to these findings, Pantoja et al. (2016) [48] in Iowa and Qin et al. (2021) [49] in Illinois reported corn grain yield reductions when corn followed CR cover crops. The contrasting results from Mississippi vs. Iowa and Illinois are actually very typical in cover-crop research due to rapid decomposition under hot and humid conditions in Mississippi vs. slow breakdown in cool and wet in Iowa and Illinois.

In 2022, low precipitation during corn critical growth period in June and July reduced N uptake (29 kg/ ha) compared to 2020 (105 kg/ha) and 2021 (101 kg/ha) (Table 7). No differences in corn grain yield were observed in 2020 and 2021 (Table 7), likely due to similar precipitation levels (Table 1) and comparable N utilizations (Table 7). These results align with findings by Spackman et al. (2019), who reported that the effectiveness of in-season nitrogen fertilization for corn is strongly influenced by growing season precipitation.

Despite corn receiving approximately equivalent nitrogen (N) rates from poultry litter and inorganic N fertilizer, corn grain yield in 2021—averaged across cover crop treatment was 20% higher with poultry litter compared to inorganic N fertilizer (9625 vs. 7666 kg/ha) (Table 7). This yield advantage associated with poultry litter in 2021 can be attributed to adequate precipitation (Table 1) and potentially enhanced N carryover [47] resulting from the mineralization of organic N from fall-applied poultry litter in 2019 and 2020. The positive effects of residual or carryover N on crop growth and yield have been well-documented [50]-[52]. Furthermore, soil amended with poultry litter typically supports more active microbial communities than those treated with inorganic fertilizer, thereby enhancing nutrient availability, improving soil health, and boosting crop productivity.

Table 7. Effects of cover crop treatments and fertilizer application on corn grain yield and grain N uptake, Plant Science Center, Mississippi State, MS.

Different letters in a column indicate a significant difference between soil amendments or cover cropping system at P≤ 0.05 level.

In 2022, averaged across cover crop treatments, no significant differences in corn grain yield were observed among fertilization treatments—including inorganic N fertilizer, broiler litter, and the unfertilized control (Table 7). This lack of response is likely due to low precipitation and limited soil moisture, which constrained nutrient mobility and uptake by plant roots [53][54]. These conditions likely increased plant stress, which collectively impeded the effectiveness of N fertilization, as evidenced by lower N uptake and reduced corn grain yields compared to 2020 and 2021 (Table 7).

The comparative yield response of unfertilized control plots, relative to poultry litter and inorganic nitrogen (N) fertilizer treatments, may be attributed to the presence of legume cover crops in control sections where no fertilizer was applied. Biological N fixation by legumes, along with N mineralization from decomposing cover crop residues, likely contributed to residual soil N accumulation in control plots, as indicated by elevated NO₃-N levels at planting (Figure 1).

A key contribution of cover cropping is nutrient conservation during fallow periods, with legumes introducing fixed nitrogen into the system. In the present study, although soil-available NO₃-N was significantly influenced by cover crops (P = 0.0105), N concentration in corn grain—which reflects soil N availability—was not significantly affected by cover crops (P = 0.0710). However, the interaction between year and cover crop was significant (P = 0.0173) (Table 6). None of the cover crop treatments, whether single species or mixtures, increased grain N content compared to the no-cover crop control in 2020 and 2021. In contrast, during the drier year of 2022, the mixed cover crop significantly increased corn grain N content relative to both single cover crops and the no-cover crop control (Table 7).

Table 8. Effects of cover crop treatments and fertilizer application on corn grain nutrient concentrations at the Plant Science Center, Mississippi State, MS (2020-2022).

Different letters in a column indicate a significant difference between soil amendments or cover cropping system at P≤ 0.05 level. Fertilizer values averaged over cover crops. Cover crop values averaged over fertilizer.

Corn grain macro- and micronutrient concentrations varied significantly across years (P < 0.0001). Interactions between year × cover crop and year × fertilization were not significant; therefore, the effects of cover crop and fertilization treatments on grain nutrient concentrations are presented as averages across years (Table 8).

Averaged across years, corn grain macro- and micronutrient concentrations—including phosphorus (P), potassium (K), sulfur (S), manganese (Mn), zinc (Zn), iron (Fe), and copper (Cu)—were not significantly affected by the main effects of cover crops (single or mixed) or fertilization treatments, with the exception of Cu. Broiler litter fertilization significantly increased Cu concentrations compared to both inorganic fertilizer and unfertilized treatments (Table 8). Given that corn grain nutrient concentrations reflect soil nutrient availability, the lack of fertilization effects on grain P and K concentrations is likely due to the very high baseline soil test P (52 mg kg⁻¹) and high soil test K (184 mg kg⁻¹) levels at the 0 - 15 cm depth, as reported by the Mississippi State University Soil Test Laboratory. Additionally, the main effects of cover crop treatments, averaged across fertilization treatments, had no significant influence on corn grain P, K, or N concentrations (Table 8).

Although fertilization treatments did not significantly affect grain nutrient concentrations, broiler litter application significantly increased grain P, K, boron (B), and Cu contents compared to the unfertilized control (Table 9). Because grain nutrient content is calculated by multiplying harvested grain yield by the corresponding nutrient concentration, the elevated grain P, K, B, and Cu contents are likely attributable to increased corn grain yield rather than differences in nutrient concentration alone.

Table 9. Effects of cover crop treatments and fertilizer application on corn grain nutrient uptake at the Plant Science Center, Mississippi State, MS (2020-2022).

Different letters in a column indicate a significant difference between soil amendments or cover cropping system at P≤ 0.05 level. Fertilizer values averaged over cover crops (main effect). Cover crop values averaged over fertilizer (main effect).

After harvesting corn, the post-harvest residual soil NO₃-N levels were significantly affected by both cover crop type and nitrogen fertilizer source. Due to a significant interaction between these factors (P = 0.0163), post-harvest NO₃-N concentrations were analyzed separately for treatments with and without cover crops (Table 10). In unfertilized control treatments, no differences in post-harvest residual NO₃-N were observed among cover crop treatments (Table 10), and the concentration of residual soil NO₃-N remained consistent across soil depths (Table 10). With inorganic N fertilizer, post-harvest soil residual NO₃-N levels were greater in no-cover crop plots at both 0 - 15 cm depth (10.3 mg/kg) and 30 - 60 cm depth (13.3 mg/kg) compared to plots with cover crops (Table 10). At 15 - 30 cm depth CC cover crop had the greatest amount of NO₃-N.

Because no-cover crop plots also received broiler litter at a rate of 4.5 mg ha⁻¹ in the fall, supplemented with inorganic N fertilizer at planting and at the V6 corn growth stage to prevent N deficiency, it appears most of the N applied at planting (56 kg/ha) leached beyond the root zone—likely a result of the underdeveloped root system early in the corn growing season. Averaged across depths, there were no significant differences in post-harvest NO₃-N between broiler litter and inorganic fertilizers at the same depth (7.3 vs. 7.0 mg/kg). However, both sources showed significantly greater NO₃-N levels compared to the unfertilized control (4.1 mg/kg) (Table 10).

Table 10. Cover crop and fertilizer interaction effect on post-harvest soil residual NO₃-N at 0 - 15-, 15 - 30- and 30 - 60 cm depth, Plant Science Center, Mississippi State, MS.

Post-harvest residual NO₃-N at 15 - 30 cm depth was substantially greater with CC cover crops (11.2 mg/kg) compared to other cover crops treatments (Table 10). This outcome may be attributed to two factors. First, CC cover crop residues decompose rapidly due to their low C/N ratio (12), potentially releasing N early in the season that the plants were unable to fully utilize, resulting in N leaching beyond the root zone. Second, excess N was released in CC plots through biological nitrogen fixation, supplemental N fertilizer at the V6 growth stage (80 kg N/ha), and fall-applied broiler litter.

With broiler litter applications, post-harvest residual NO₃-N accumulation was observed only in CC plots and in plots with mixed cover crops at 0 - 15 cm depth (Table 10). Beyond 15 cm depth, no differences in post-harvest residual NO₃-N were found among cover crop treatments with broiler litter applications. This is likely due to the slow N release from residues, which may have been synchronized with corn N uptake.

Averaged across depths, post-harvest residual NO₃-N did not differ between broiler litter (7.0 mg/kg) and inorganic N fertilizer (7.3 mg/kg), though both were approximately 44% greater than the unfertilized control (4.1 mg/kg) (Table 10). Across years, no differences in corn grain yield were observed between the control and inorganic N fertilizer treatments (Table 7). Interestingly, averaged across fertilization treatments, post-harvest residual NO₃-N did not differ among cover crop treatments, except at the 0 - 15 cm depth, where CC plots had significantly greater NO₃-N than CR plots (Table 10).