Gypsum, crop rotation, and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems.

Soil organic carbon (SOC), a core soil quality indicator, is influenced by management practices. The objective of our 2012-2016 study was to elucidate the impact of gypsum, crop rotation, and cover crop on SOC and several of its biological indicators under no-till in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) in the USA. A randomized complete block design in factorial arrangement with gypsum (at 0, 1.1, and 2.2 Mg/ha annually), rye (Secale cereal L.) vs no cover crop, and rotation (continuous soybean [Glycine max (L) Merr., SS] vs corn [Zea mays, L.]-soybean, both the CS and SC phases) was conducted. Composite soils were collected (0-15 cm and 15-30 cm) in 2016 to analyze microbial biomass C (SMBC), SOC, total N, active C, cold and hot-water extractable C, C and N pool indices (CPI and NPI), and C management index (CMI). Results varied for main effects of gypsum, crop rotation, and cover crop on SOC pools, total N, and SOC lability within and across the sites. Gypsum at 2.2 Mg/ha increased SMBC within sites and by 41% averaged across sites. Likewise, gypsum increased SMBC:SOC, active C, and hot-water C (as indicators of labile SOC) averaged across sites. CS rotation increased SOC, active C, CPI, and CMI compared to SS, but decreased SMBC and SMBC:SOC within and across sites. CPI had a significant relationship with NPI across all sites (R2 = 0.90). Management sensitive SOC pools that responded to the combined gypsum (2.2 Mg/ha), crop rotation (CS), and cover crop (rye) were SMBC, SMBC:SOC, active C, and CMI via SMBC. These variables can provide an early indication of management-induced changes in SOC storage and its lability. Our results show that when SOC accumulates, its lability has decreased, presumably because the SMBC has processed all readily available C into a less labile form.

Introduction

Soil organic carbon (SOC) consists of diverse organic compounds that affect soil and environmental functions [1-4]. It is one of the core indicators of soil quality, and acts as a reservoir of substrates, energy, enzymes, and nutrients that maintains biodiversity and efficiency, regulates biochemical reactions and buffering, and provides soil physical stability [5-7]. However, considering SOC as one homogeneous pool ignores variations in the chemical composition and relative proportion of its individual pools, and how these SOC pools both influence and reflect agricultural management practices [8].

The various SOC pools are diverse in terms of their chemical composition, lability and biochemical turnover, and physico-chemical stability [1, 9, 10]. Because a detectable change in the SOC often takes several years, researchers have, therefore, emphasized the need to measure labile SOC pools, thus providing early indications of management-induced changes in SOC content associated with soil quality [5, 8, 11].

Labile C is defined as a small pool of SOC that has rapid turnover rates and is preferably biochemically utilized compared to the rest of the SOC [7, 8, 12]. The SOC pools that are considered as labile are active C, SMBC, potentially mineralizable C, light-fraction, cold and hot-water extractable C, anthrone reactive C, particulate organic C, and beta-glucosidase activity [5, 11–14]. Several metabolic quotients and indices such as basal respiration, SMBC:SOC, microbial cell death rates, specific maintenance respiration rates, and C and N management indices (CMI and NMI) have been suggested as sensitive indicators of SOC and TN dynamics [4, 14–16]. While the amount of C associated with SMBC is small, it is considered a key labile biological component that catalyzes SOC and N transformations. Hence, it is considered of prime importance for labile SOC utilization and nutrient cycling in soil [4, 17].

Management practices of repeated annual plowing, unbalanced and excessive chemical fertilization, and continuous monocropping have led to rapid depletion of labile SOC pools. Early detection of temporal changes from labile to non-labile SOC pools, or vice-versa, caused by agricultural management practices can be useful in helping producers change their practices before they become difficult to reverse and remediate. Agricultural management practices that diversify and maintain a balance between SOC accumulation and lability can improve soil quality and support economic crop productivity. An adoption of sustainable agricultural practices (i.e., continuous no-till, crop rotation with cover crops, residue management, precision fertilization, and soil amendments) is expected to improve soil’s functional capacity and lead to enhanced agroecosystem services [7, 16].

Recently, gypsum, which is hydrated calcium sulfate (CaSO4•2H2O) mineral from mined geologic deposits, a co-product of the phosphate fertilizer industries, or a byproduct of flue gas desulfurization of coal-based power plants, has received increased attention as an alternate source of Ca and S for plant nutrition, a proactive chemical amendment for environmental remediation, and for soil quality improvement [16, 18–21]. Gypsum is widely used as a chemical amendment to reclaim sodic and marginal soils for crop production, a conditioner of soil physical properties to reduce surface crusts and improve soil structures, an electrolyte to improve water infiltration, and a treatment to reduce edge-of-field loss of soluble reactive phosphorus [22, 23]. Recently, a few studies have reported that gypsum application favors soil biology and enzymatic activity in Hagerstown silt loam (pH 6.2) in Pennsylvania, USA [16] and in alkaline-saline clay soils in northwest China [24]. Gypsum amendments reportedly provide several nutrients essential for crop growth in Marvyn loamy sand in Alabama (Shorter), Blount silt loam in Indiana (Farmland), Hoytville clay in northwestern Ohio, and Omulga silt loam in southern Ohio, respectively [21].

Gypsum can play a critical role in regulating SOC transformations due to its high concentration of Ca [18, 21]. The cationic bridging effects of Ca promote flocculation of clay and SOC in the formation of water-stable aggregates that lead to the physical accumulation of SOC as particulate organic C [16, 18]. In contrast, several other studies report that Ca-based amendments, such as lime and gypsum, may lead to SOC depletion due to increased activity of opportunistic microbes via a priming effect on native SOC content [24]. While research on gypsum amendments has primarily focused on improving or restoring physical and chemical properties of sodic and marginal soils, more information is needed to elucidate its effects on soil biology and SOC pools under agronomic cropping diversity.

The cropping diversity that currently dominates the Midwest United States is the corn-soybean (CS) rotation. The success of annually plowed soil that often accompanies the CS rotation has not come without environmental costs and has resulted in SOC depletion over time [25]. Crop rotation, when combined with conservation tillage, especially no-till, is a biological diversity management option to improve soil functionality [25, 26]. It provides several agroecosystem services such as diverse ground covers to shorten fallow periods, minimize soil erosion, suppress weed and disease infestations, improve hydraulic conductivity, and increase soil aggregate formation with an associated decrease in soil crusting, sealing, and compaction [26-28].

Likewise, cover crops, when included into crop rotations, provide extended ground cover, support rhizosphere effects, and exert synergistic effects on SMBC and biological activities, thereby promoting root growth that improves soil aeration and drainage. Cover crops also serve as a source of diverse and biochemically labile C and essential nutrients, phytochemicals, and extracellular enzymes, leading to efficient nutrient recycling and increased SOC accumulation [29]. In recent years, there has been increased interest in integrating cereal rye as a winter hardy cover crop within CS rotations [28-30]. Rye quickly resumes growth in the early spring from winter dormancy and produces a substantial amount of biomass across a broad range of site conditions. By adding surface and subsurface biomass inputs, rye aids in soil aggregate formation that leads to physical protection of SOC, improves soil quality, and increases crop productivity resilience to climate change events [16, 31].

Considering the close relationships among SOC and soil biological, chemical, and physical properties and processes [4, 8, 16], information on agricultural management-induced effects related to early changes in SOC accumulation and lability is important to evaluate soil quality. However, long-term effects of gypsum, crop rotation, and cover crop on soil biology, total N, and SOC pools, under a rainfed no-till system across different sites and weather conditions, but under similar management practices, remain mostly unexplored.

We hypothesize that the integration of gypsum in CS rotations with rye as a winter cover crop will provide diverse organic inputs that create favorable site conditions for increased biological efficiency, and thereby potentially influence the SOC pools across geographic locations. A five-year field research study was conducted under diverse soil and climatic conditions of the Midwest and Southeastern United States with an objective to evaluate how gypsum and cereal rye under different phases of CS rotation impact soil biology, total N, and SOC pools in a rainfed, transitional no-till system.

Materials and methods

Site description

No-till field experiments were conducted simultaneously at four different sites under rainfed conditions across different soil types and climate zones (Fig 1) that included Alabama (Agricultural Experiment Station’s E.V. Smith Research Center-Field Crops Unit, 32°25’19” N, 85°53’7” W at Shorter), Indiana (Davis Purdue Agricultural Center near Muncie (40°15’34.7" N 85°09’19.9" W at Farmland), and Ohio (Northwest Agricultural Research Station, Ohio Agricultural Research & Development Center, 41°13’18.26” N, 83°45’34.91” W at Hoytville and Ohio State University South Centers research farm, 39°06’78” N, 83°00’92” W at Piketon) in the U.S. Midwest [21, 32, 33].

Fig 1

Gypsum, crop rotation and cover crop rainfed field experiments at Alabama (pictures depict crimper rolling of rye winter cover crop followed by plating of corn and soybeans, and matured corn-soybeans before harvesting), Indiana (pictures depict rye as a winter cover crop followed by growing of soybeans within rye mulch, and soybeans at maximum vegetative growth stage), Ohio-north (pictures depict rye as a winter cover crop in early and late spring before planting of corn, and corn at early maturity stage), and Ohio-south (pictures depict rye as a winter cover crop followed by growing of corn within rye residues, and collected soil cores in plastic tubes using the JMC® stainless-steel soil environmental probe; https://www.jmcsoil.com) in the U.S. Midwest during 2012 to 2016 (Photos taken by Randall C. Reeder and Khandakar R. Islam).

Soil at the Alabama site (Shorter) is a deep, well-drained, and moderately permeable Marvyn loamy sand (fine-loamy, kaolinitic, thermic Typic Kanhapludult). Initial analysis showed soil at the 0–15 cm and 15–30 cm depths, respectively, had values of pH 5.9 and 5.5, electrical conductivity (ECe) 170 and 101 μS/cm, SOC 0.39 and 0.30%, total N 0.032 and 0.028%, active C 111 and 79 mg/kg, extractable Ca 0.39 and 0.32 g/kg, extractable sulfur 0.50 and 0.56 g/kg, and bulk density (ρb) 1.53 and 1.62 g/cm.

In contrast, soil at the Indiana site (Farmland) is an association of poorly drained Blount silt loam (fine, illitic, mesic Aeric Epiaqualfs), Glynwood silt loam (fine, illitic, mesic Aquic Hapludalfs), and Pewamo silty clay loam (fine, mixed, active, mesic Fluvaquentic Endoaquolls). Soil at the 0–15 cm and 15–30 cm depths, respectively, had values of pH 6.0 and 6.1, ECe 290 and 173 μS/cm, SOC 1.22 and 0.97%, total N 0.105 and 0.090%, active C 347 and 294 mg/kg, extractable Ca 1.76 and 1.45 g/kg, extractable sulfur 0.38 and 0.40 g/kg, and ρb 1.25 and 1.29 g/cm3.

Soil at the Ohio north site (Hoytville) is a poorly drained Hoytville clay (fine, illitic, mesic Mollic Epiaqualfs). Soil at the 0–15 cm and 15–30 cm depths, respectively, had values of pH 6.2 and 6.3, ECe 315 and 278 μS/cm, SOC 1.93 and 1.82%, total N 0.213 and 0.193%, active C 407 and 303 mg/kg, extractable Ca 4.32 and 4.72 g/kg, extractable sulfur 0.41 and 0.41 g/kg, and ρb 1.32 and 1.39 g/cm3. In contrast to the Ohio north site, soil at Ohio south site is a moderately well-drained Omulga silt loam (fine-silty, mixed, active, mesic Oxyaquic Fragiudalfs). Soil at the 0–15 cm and 15–30 cm depths, respectively, at this site had values pH 6.1 and 5.9, ECe 88 and 66 μS/cm, SOC 0.59 and 0.33%, total N 0.060 and 0.032%, active C 262 and 107 mg/kg, extractable Ca 1.05 and 0.86 g/kg, extractable sulfur 0.20 and 0.15 g/kg, and ρb 1.41 and 1.62 g/cm3 [21].

Experimental design and cultural practices

Prior to establishing the experiment, all sites were under some type of tillage operations and a CS rotation without any cover crops or gypsum amendments. A three-factorial experiment (3 gypsum rates x 2 cover crops x 3 crop rotations) in a randomized complete block design, with four replications for each treatment combination, was established at all locations in the autumn of 2012 [21, 33]. The gypsum rates were 0, 1.1, and 2.2 Mg/ha, which were annually surface-applied to the transitioning no-till, except at the Ohio south site, where gypsum was applied only in the fall of 2012 and 2014. Crop rotation included continuous SS versus both phases of CS rotation each year with cereal rye as a cover crop versus no cover crop. Cereal rye at the rate of 60 kg/ha was drilled into soil after harvesting of corn or soybeans in October during the experimental period.

Agronomic practices required by the experimental design were managed commensurate with local weather, soil, and pest and weed pressure conditions [21, 32]. All management practices, with the exception of the treatments applied, were performed as similar as possible across all treatments in regard to the geographic, climatic, and time scales. Furthermore, agronomic cultural practices and cover crop planting were performed accordingly. Cover crop was terminated with crimper roller and Roundup® approximately two weeks before soybean or corn planting (Fig 1).

Soil sampling and analysis

Soil cores (2.54 cm internal dia.) were extracted using the JMC® stainless-steel soil environmental probe lining with plastic tube immediately after crop harvest in October 2016 from geo-referenced sites within each replicated plot at 0–15 cm and 15–30 cm depth intervals. A minimum of three field-moist soil cores were composited for each replicated plot and placed in sealable plastic bags for temporary storage at 4°C until needed. A portion of the field-moist soil was gently sieved through a 2-mm mesh prior for measuring SMBC. Another portion of the field-moist soil was air-dried for a period of approximately 15 days under shade at room temperature, processed, and analyzed for chemical and physical properties [21].

The SMBC was measured by a rapid microwave irradiation and extraction method [34] with the determination of total extracted organic C in an extract aliquot using a Shimadzu® total dissolved SOC/TN analyzer. The SMBC was calculated as follows, where the CMW and CUMW represent extracted C in microwaved and unmicrowaved soils, and the KME is the fraction of the SMBC (0.213) extracted by 0.5M K2SO4.

The metabolic quotient (SMBC:SOC) was calculated by dividing the SMBC by the SOC [15]. The SOC and total N were determined on finely ground (< 125 μm), air-dried soil by the dry combustion method using a FlashEA-1112 series CNHS-O analyzer®. Active C was determined spectrophotometrically on a separate soil sample after a mild potassium permanganate oxidation (0.02 M at pH 7) of air-dried soil [8]. Both cold and hot-water C were also extracted on separate soil samples and determined using a Shimadzu® total dissolved SOC/TN analyzer [11, 16].

Basic soil chemical and physical properties such as pH (1:2 in soil and distilled deionized water suspension) by glass electrode, ECe (1: 1 soil and distilled deionized water paste) by electrical conductivity probe, and ρb by standard core methods were determined.

Soil organic carbon and nitrogen lability and management indices

Using the measured SOC, active C, SMBC, and cold and hot-water C data, the C management index (CMI) was calculated as follows [13, 16]: where the CPI is the C pool index and the CLI is the C lability index, which were calculated as: where CL refers to the lability of C, which was calculated as:

Using the same principle, the nitrogen pool index (NPI) was calculated as follows [16]:

The labile C pool was considered as a portion of the SOC pool that was comprised of active C, SMBC, or cold and hot-water C. The non-labile C pool was calculated by subtracting the active C, SMBC, or cold and hot-water C pool from the SOC [16].

Statistical analysis

Multivariate statistical analyses were performed by using SAS® on the measured and calculated soil properties attributed to the impacts of gypsum application, crop rotation, cover crop, and their interactive effects for each experimental site [35]. While gypsum, crop rotation, and cover crop were considered as fixed variables, block was considered as a random variable to perform analysis of variance (ANOVA). Moreover, to evaluate the impacts of gypsum, crop rotation, and cover crop across all the replicated research sites, a meta-analysis was performed [35, 36]. To conduct a meta-analysis of the pooled data from all sites, first data were normalized by dividing the treatment values of a variable with the control value of that same variable at each site to provide a ratio. The controls were the zero rate of gypsum application, the CS rotation, and the minus cover crop treatment. The normalized ratios were then statistically analyzed using the ANOVA procedures as described above to evaluate gypsum, crop rotation, and cover crop main effects, and their interaction effects on SMBC, SOC pools, TN, CPI, and CMI indices across all sites. For all statistical analyses, the significant main effect and interactions of treatment variables on soil properties were separated by the F-protected Least Significant Difference (LSD) test with a value of p ≤ 0.05 unless otherwise mentioned. Regression and correlation analyses were performed using SigmaPlot®.

Results and discussion

Significant but variable main effects of gypsum application, crop rotation, and cover crop on soil properties such as SMBC, SOC pools, total N, and SOC lability indices without consistent interactive effects among and across the sites were observed (Tables 1–5). Accordingly, the main effects of gypsum application, crop rotation, and cover crop on soil properties are discussed (Tables 1–5).

Table 1

Gypsum and cover crop impacts on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBMC), active carbon (AC), cold (CWC) and hot (HWS) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI), and carbon management index (CMI) under different phases of a rainfed no-till soybean-corn rotation in Alabama (2012 to 2016).

Treatment	Variable response
	SOC	TN	SMBC	SMBC:	AC	CWC	HWC	CPI	NPI	CLI				CMI
Gypsum (Mg/ha)	(g/kg)		(mg/kg)	SOC (%)	(mg/kg)					SMBC	AC	CWC	HWC	SMBC	AC	CWC	HWC
0	6.3a¥	0.53ab	103b	2.05ab	182b	10.2a	30.2b	1.00a	1.00b	1.06b	1.01a	1.00a	0.96a	1.06b	1.01a	1.00a	0.96a
1.1	6.4a	0.50b	119ab	2.46a	192ab	12.4a	36.2ab	1.01a	0.94c	1.08ab	1.02a	0.99a	0.98a	1.09ab	1.03a	1.00a	0.99a
2.2	6.5a	0.61a	138a	2.5a	221a	10.5a	38.1a	1.03a	1.15a	1.16a	1.07a	1.01a	1.03a	1.19a	1.10a	1.04a	1.06a
Crop rotation
CS	7.7A≠	0.53A	122A	1.75B	247A	11.5A	35.2A	1.23A	1.00A	1.18A	1.09A	1.04A	1.05A	1.45A	1.34A	1.28A	1.29A
SC	7A	0.55A	111A	1.81B	203A	10.2A	31.5A	1.08B	1.04A	1.23A	1.12A	1.08A	1.10A	1.33B	1.21B	1.17B	1.19B
SS	4.7B	0.56A	128A	3.44A	144B	11.4A	37.7A	0.74C	1.06A	1.26A	1.13A	1.10A	1.13A	0.93C	0.84C	0.81C	0.84C
Cover crop
Control	6.3x£	0.50x	118x	2.27x	195x	11.1x	35.6x	1.00x	0.95y	1.29x	1.12x	1.16x	1.15x	1.29x	1.12x	1.16x	1.15x
Rye	6.5x	0.60x	123x	2.40x	202x	10.9x	34.0x	1.03x	1.11x	1.28x	1.11x	1.17x	1.16x	1.32x	1.14x	1.21x	1.19x

¥ Means separated by same lower-case letter (a, b, and c) in each column were nonsignificant (at p≤0.05) among gypsum application rates by the LSD test.

≠ Means separated by same upper-case letter (A, B and C) in each column were nonsignificant (at p≤0.05) among crop rotations by the LSD test.

£ Means separated by same lower-case letter (x and y) in each column were nonsignificant (at p≤0.05) between cover crops by the LSD test.

Table 5

Gypsum and cover crop impacts on normalized values of total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBMC), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under different phases of a rainfed no-till soybean-corn rotation when averaged across all sites (2012 to 2016).

Treatment	Variable response
	SOC	TN	SMBC	SMBC:	AC	CWC	HWC	CPI	NPI	CLI				CMI
Gypsum (Mg/ha)	(g/kg)		(mg/kg)	SOC (%)	(mg/kg)					SMBC	AC	CWC	HWC	SMBC	AC	CWC	HWC
0	1.01a¥	1.00a	0.96c	0.96c	1.02b	1.02a	0.97c	1.00a	1.00a	1.01a	1.03a	1.02a	0.96a	1.00a	0.98a	1.00a	1.00a
1.1	1.02a	0.99a	1.13b	1.16b	1.06ab	1.11a	1.08b	1.01a	0.99a	1.05a	1.03a	1.02a	0.98a	1.06a	1.00a	1.02a	1.04a
2.2	1.03a	1.05a	1.37c	1.32a	1.15a	1.10a	1.20a	1.02a	1.06a	1.08a	1.04a	1.02a	1.00a	1.07a	1.03a	1.05a	1.07a
Crop rotation
CS	1.14A≠	1.05A	1.12B	0.96C	1.21A	1.09A	1.08A	1.12A	1.06A	1.10A	1.05A	1.03A	1.01A	1.24A	1.19A	1.21A	1.25A
SC	1.00AB	0.99A	1.12B	1.08B	1.07AB	1.05A	1.05A	1.01B	0.99A	1.12A	1.06A	1.06A	1.03A	1.03B	1.00B	1.04B	1.06B
SS	0.92B	0.99A	1.22A	1.40A	0.94B	1.09A	1.11A	0.91B	0.99A	1.14A	1.06A	1.07A	1.04A	0.95B	0.89C	0.93C	0.96C
Cover crop
Control	1.01x£	0.99x	1.13x	1.13x	1.05x	1.06x	1.06x	1.01x	1.00x	1.19x	1.07x	1.10x	1.07x	1.11x	1.03x	1.10x	1.12x
Rye	1.03x	1.03x	1.18x	1.16x	1.10x	1.09x	1.10x	1.02x	1.04x	1.21x	1.06x	1.09x	1.08x	1.14x	1.04x	1.10x	1.14x

Means separated by same lower-case letter (a, b, and c) in each column were nonsignificant (at p≤0.05) among gypsum application rates by the LSD test.

≠ Means separated by same upper-case letter (A, B and C) in each column were nonsignificant (at p≤0.05) among crop rotations by the LSD test.

Means separated by same lower-case letter (x and y) in each column were nonsignificant (at p≤0.05) between cover crops by the LSD test.

Gypsum impacts on soil carbon pools and lability

While the SOC and total N were unaffected, the labile SOC pools were variably influenced by gypsum application rates (Tables 1–5). In this section, all comparisons are made in reference to the 0-gypsum rate treatment unless noted otherwise. The SMBC, as a biological component of the labile SOC pool, was increased with gypsum treatment at all sites (25 to 33%) with the increase being greater for the 2.2 versus the 1.1 Mg/ha gypsum treatment. The size of the biological C pool (SMBC:SOC) also increased with gypsum application at all sites except Ohio-Piketon. Gypsum applied at the 2.2 kg/ha significantly increased both active C and hot-water C pools.

The CPI, (i.e., a measure of SOC accumulation or depletion) in general, did not change significantly. The NPI value, however, was increased significantly, but only in Alabama (by 15%) by the 2.2 Mg/ha gypsum application (Table 1). The SOC lability did not vary consistently except for a slight improvement (by 8 to 10%), due to a change in the SMBC value in Alabama, where the 2.2 Mg gypsum/ha was applied (Table 1). In contrast, the CMI, a composite measure of SOC accumulation and lability, was significantly affected (by 12 to 13%) via a change in SMBC by the impact of the 2.2 Mg/ha gypsum application in both Alabama and Ohio-Hoytville sites.

When the data on SOC pools, total N, and SOC lability were normalized at each site, followed by an across-sites analyses (i.e., a meta-analysis), SMBC (by 41%), SMBC:SOC (by 36%), active C (by 13%), and hot-water C (by 23%) showed positive responses to the 2.2 Mg/ha gypsum, but without any significant increase in SOC accumulation or lability (Table 5).

A significant increase in the SMBC, SMBC:SOC, active C, and hot-water C indicate a biochemical response to the gypsum amendments that contributes to improved soil quality. Gypsum is more soluble (by about 150 to 200 times) than lime, another Ca-containing amendment that increases the mobility of Ca+2, Mg+2, and K+ associated with SO4-2 in the soil profile [19, 21]. This influences soil chemical and physical properties that are more favorable for SMBC and associated biological activities [16]. Other studies, however, have reported that soil biological and chemical properties were not consistently influenced by gypsum or similar types of soil chemical amendments [24, 37].

While the SMBC is only a small fraction (~ 5%) of SOC, it acts as a proactive biocatalyst for residue decomposition, nutrient recycling, and aggregate formation associated with soil quality [5, 38]. An increase in SMBC translates to an enlargement in the size of the biologically labile pool of SOC (SMBC:SOC), which shows a positive response with improved C-use efficiency towards SOC accumulation and lability [4, 5, 15].

Several studies have reported that the SMBC and community structures act indirectly and positively to the changing or balancing of soil geochemical and physical properties by agricultural management practices [24, 39]. Gypsum amendments combined with transitional no-till seem to affect the SMBC diversity, including fungi dominance [39]; however, as reported, not all microbes are sensitive to gypsum application [24]. Our results showed that gypsum, applied at the 2.2 Mg/ha rate, exerted complementary effects on the no-till performance to increase SMBC and the size of the biologically labile pool of SOC (SMBC:SOC). This is thought to be primarily mediated by shifting a change towards a fungi-based food web and an improvement in soil functions under no-till [16, 17, 39]. Likewise, significantly higher active C and hot-water C were observed due to synergistic effects of microbes-residue interactions, including both agronomic and cover crops’ response to gypsum application. A significantly higher SMBC with improved C-use efficiency (higher SMBC:SOC) may have acted positively to affect a higher level of formation of active C and hot-water C via a change in SMBC [8, 11, 15]. This is attributed to a greater release or formation of microbial metabolites and root exudates, and production of fine roots of both agronomic and cover crops as influenced by Ca, Mg, and S nutrition from the gypsum amendments [9, 16]. As the SMBC and SMBC:SOC values were higher in the 2.2 Mg/ha gypsum rate, this indicates the prolonged and cumulative residual impact of gypsum applied as a proactive soil chemical amendment.

Significantly higher values of CMI in the marginal soils of Alabama suggests that a change is occurring in SOC lability, but not in significant accumulation. This was mostly attributed to an increase in the size of the SMBC pool and its efficient C anabolism. Soils with higher CMI values are considered to be better managed with higher C-use efficiency towards improved soil quality [16, 40]. In this study, a consistent effect of the 2.2 Mg/ha gypsum application rate on SMBC, SMBC:SOC, and CMI was observed when meta-analysis of data was performed across the sites.

Crop rotation impacts on soil carbon pools and lability

The impact of CS rotation in different phases compared to SS variably affected the SOC pools and total N (Tables 1–5). The SOC values were higher by 49 to 60% under both CS phases than under SS in Alabama, and were also higher, to a lesser degree, under CS-S and SS rotations, respectively, in Indiana and Ohio-Piketon. Total N was also significantly higher in Indiana by more than 12% in soils under CS-C than in soils under both CS-S and SS rotations.

The SMBC values, in contrast, were significantly higher in Indiana under SS than under both phases of the CS rotation, but significantly lower in Ohio-Piketon under SS than under both CS phases (Tables 4 and 5). A positive impact of SS compared to CS phases on SMBC:SOC was also observed at all sites except Ohio-Hoytville. Like SMBC, the active C values were similarly greater under both CS phases than under SS at all sites except Indiana. Crop rotation also variably influenced hot-water C.

Table 4

Gypsum and cover crop impacts on total soil organic C (SOC), total nitrogen, microbial biomass (SBMC), active carbon (AC), cold (CWC) and hot (HWC) salt solution extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under different phases of a rainfed no-till soybean-corn rotation in Ohio-Piketon (2012 to 2016).

Treatment	Variable response
	SOC	TN	SMBC	SMBC:	AC	CWC	HWC	CPI	NPI	CLI				CMI
Gypsum (Mg/ha)	(g/kg)		(mg/kg)	SOC (%)	(mg/kg)					SMBC	AC	CWC	HWC	SMBC	AC	CWC	HWC
0	6.9a¥	0.86a	205a	3.19ab	338b	15.1a	58.7a	1.00a	1.00a	1.00a	1.02a	0.96a	0.89a	1.00a	1.02a	0.96a	0.89a
1.1	6.9a	0.85a	201a	3.39a	368ab	14.7a	57.6a	1.02a	1.01a	1.04a	1.02a	0.96a	0.87a	1.05a	1.04a	0.98a	0.88a
2.2	7a	0.88a	204a	3.01b	418a	15.8a	59.3a	1.03a	1.04a	1.04a	1.03a	0.97a	0.87a	1.07a	1.06a	1.00a	0.90a
Crop rotation
CS	7.7A≠	0.91A	202A	2.7B	440A	14.9A	57.9AB	1.11A	1.09A	1.04A	1.02A	0.96A	0.87A	1.16A	1.14A	1.07A	0.97A
SC	6.7B	0.83A	232A	3.50A	391AB	15.7A	65.2A	1.00B	0.99B	1.04A	1.04A	0.97A	0.87A	1.04B	1.04B	0.97B	0.87B
SS	6.4B	0.83A	177B	3.38A	293B	15A	52.6B	0.93B	0.99B	1.01A	1.02A	0.96A	0.86A	0.95B	0.95B	0.90B	0.80B
Cover crop
Control	6.9x£	0.87x	193x	3.05y	370x	15.2x	61.0x	1.02x	1.01x	1.01x	1.05x	0.98x	0.86x	1.03x	1.07x	1.00x	0.87x
Rye	6.9x	0.85x	215x	3.35x	380x	15.2x	56.2x	1.02x	1.03x	1.01x	1.04x	0.95x	0.85x	1.03x	1.06x	0.97x	0.86x

¥ Means separated by same lower-case letter (a, b, and c) in each column were nonsignificant (at p≤0.05) among gypsum application rates by the LSD test.

≠ Means separated by same upper-case letter (A, B and C) in each column were nonsignificant (at p≤0.05) among crop rotations by the LSD test.

£ Means separated by same lower-case letter (x and y) in each column were nonsignificant (at p≤0.05) between cover crops by the LSD test.

The CPI was significantly higher by 11 to 49% at all sites except Ohio-Hoytville under both CS phases when compared with SS (Tables 1, 3 and 4). In contrast, the NPI was significantly higher in Indiana (by 19 to 22%) and Ohio-Piketon (by 10%) under both CS phases compared to SS. The CMI, in contrast, did vary significantly by the impact of crop rotation at all sites. The CMI, as influenced by SMBC, was higher under both CS phases compared to SS at all sites except Ohio-Hoytville, where an opposite trend was observed. The CMI values, as influenced via active C and cold and hot-water C, had similarly higher values under both CS phases than SS at all sites except Ohio-Hoytville.

Table 3

Gypsum and cover crop impacts on total soil organic C (SOC), total nitrogen, microbial biomass (SBMC), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under different phases of a rainfed no-till soybean-corn rotation in Indiana (2012 to 2016).

Treatment	Variable response
	SOC	TN	SMBC	SMBC:	AC	CWC	HWC	CPI	NPI	CLI				CMI
Gypsum (Mg/ha)	(g/kg)		(mg/kg)	SOC (%)	(mg/kg)					SMBC	AC	CWC	HWC	SMBC	AC	CWC	HWC
0	12.7a¥	1.39a	143c	1.23c	405a	27.8a	40.0b	0.99a	1.00a	0.99a	1.07a	1.05a	0.98a	0.98a	1.06a	1.04a	0.97a
1.1	12.4a	1.38a	211b	1.84b	403a	26.8a	44.8b	0.96a	0.99a	1.04a	1.07a	1.05a	1.01a	1.00a	1.03a	1.01a	0.97a
2.2	12.6a	1.41a	296a	2.49a	405a	31.1a	56.3a	0.98a	1.01a	1.08a	1.07a	1.04a	1.02a	1.06a	1.05a	1.02a	1.00a
Crop rotation
CS	14.4A≠	1.58A	193B	1.42C	455A	28.6A	45B	1.12A	1.14A	1.08A	1.07A	1.02A	1.02A	1.21A	1.20A	1.14A	1.14A
SC	11.7B	1.32B	199B	1.8B	365B	27.2A	44.1B	0.91B	0.95B	1.05A	1.06A	1.06A	1.01A	0.96B	0.96B	0.96A	0.92B
SS	11.6B	1.29B	259A	2.33A	393B	29.9A	52.0A	0.90B	0.92B	1.06A	1.06A	1.10A	1.03A	0.95B	0.95B	0.99A	0.93B
Cover crop
Control	12.5x£	1.41x	203y	1.80x	404x	28x	45.3x	0.97x	1.00x	1.06x	1.06x	1.12x	1.05x	1.03x	1.03x	1.09x	1.02x
Rye	12.7x	1.40x	231x	1.91x	404x	29.1x	48.8x	0.98x	1.00x	1.10x	1.07x	1.12x	1.07x	1.08x	1.05x	1.10x	1.05x

¥ Means separated by same lower-case letter (a, b, and c) in each column were nonsignificant (at p≤0.05) among gypsum application rates by the LSD test.

≠ Means separated by same upper-case letter (A, B and C) in each column were nonsignificant (at p≤0.05) among crop rotations by the LSD test.

£ Means separated by same lower-case letter (x and y) in each column were nonsignificant (at p≤0.05) between cover crops by the LSD test.

When the data were normalized at each site, followed by across the sites (i.e., meta-analysis), the SOC, active C, CPI, and CMIs values increased under both CS phases when compared to SS. An opposite trend, however, was observed on SMBC and SMBC:SOC by crop rotation (Table 5). The values of CMIs via a change in SMBC was consistently higher than active C and cold and hot-water C under both CS phases when compared to SS.

In agroecosystems, the primary input of SOC is shoot and root biomass of crops. An increase in SOC across the sites, except Ohio-Hoytville, under CS-C compared to SS was due to increased deposition of C inputs that consists of larger amounts of C-enriched corn biomass followed by lesser amounts of highly decomposable N-rich soybean biomass. In comparison, there was much less total C input under SS. Crop residues with a high C:N ratio that are returned to soil are expected to decompose more slowly and have a longer residence time in marginal soils like Alabama and Ohio-Piketon [9, 25, 41]. Furthermore, the combination between residue retention and transitional no-till has a greater impact in accumulating SOC under CS-C than under SS.

A variability in SMBC and SMBC:SOC values caused by crop rotations at different sites suggests a proportionally variable shift in the biologically labile pool of SOC as influenced by the amount and diversity of residue C and nutrients returning to the soil. An increase in SMBC and SMBC:SOC across the experimental sites over time was due to surface accumulation of C-enriched residue from corn and N-enriched residue from soybeans [25, 26, 42]. Moreover, inherent regional and site variability such as climatic conditions, geochemical, soil types and texture, and moisture dynamics are also thought to have influenced the variability in SMBC and SMBC:SOC values [5, 34]. However, several studies have suggested that the effects of residue quality are mostly short-term, and that all residues become chemically similar once processed by SMBC [42]. This has been supported by the emerging new concepts of SOC formation, which suggest that the labile pools of SOC are composed mostly of microbial cells and their metabolites [9, 42].

Significant increase in active C values under both CS phases was also associated with the greater amount of corn and soybean residues returning to the soil than the smaller amount of soybean residues over time under SS. The active C values, a measure of labile SOC, is closely associated with SOC and SMBC, and an increase or a depletion in SOC and SMBC is also expected to invariably affect the active C content [8, 12, 16]. However, as previously stated, the amount and quality of residue returning, and regional and site variability, are also expected to play important roles. Given the critical impact that SMBC has in processing crop residues, and indirectly contributing their cells and their metabolites to active C and SOC formation [9, 16, 43], the positive effects of crop rotation may have led to greater SOC accumulation in Alabama and Ohio-Piketon soils than in Ohio-Hoytville and Indiana soils. This was reflected in the higher CMI values via CPI observed for the CS rotations.

The highest CMIs observed under both CS phases followed by SS across the sites were attributed to the higher CPI values that were impacted by the greater amounts of C released upon decomposition of diverse crop residues as microbial cells and metabolites, fine roots production, root exudates, and humus. Because CMI is an integrated measure for both the quantitative and qualitative nature of SOC, soils with higher CMIs, as influenced by greater availability of labile C, are considered to be better managed to improve soil quality [4, 16, 40]. The CMI may induce changes in other soil functional properties including biodiversity and efficiency, enzyme activities, and N availability in response to balance among microbes and plants, residue decomposition, and SOM formation and mineralization.

In our study, the changes observed in CMI values, as induced by agricultural management practices, were of a higher degree of CPI than changes on SOC lability (CLI), a tendency also observed in other studies [4, 16, 40]. In other words, the CPI values control the values of the CMI. Thus, calculating CPI and CMI values provide a way to detect early changes in SOC lability and accumulation or depletion, which are undetectable when considering SOC values.

Cover crop impacts on soil carbon pools and lability

Integrating rye as a winter cover crop into the crop rotation exerted limited impacts on SOC pools, total N, and SOC lability (Tables 1–5). Rye significantly increased the SMBC (by 14 to 24%) in Ohio-Hoytville and Indiana soils, respectively, when compared to their control soils. The SMBC:SOC was also significantly increased by the impact of rye in Ohio-Hoytville and Ohio-Piketon. Likewise, the active C was increased by the rye at all sites, but only significantly at Ohio-Hoytville (Table 2). While the CPI did not change, the NPI values were increased (by 16%) under rye, but only in Alabama (Table 1). Normalization of data among and across the sites showed that rye promoted only a slight increase in SOC pools, total N, and SOC lability, and this increase was not consistently significant (Table 5).

Table 2

Gypsum and cover crop impacts on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBMC), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI), and carbon management index (CMI) under different phases of a rainfed no-till soybean-corn rotation in Ohio-Hoytville (2012 to 2016).

Treatment	Variable response
	SOC	TN	SMBC	SMBC:	AC	CWC	HWC	CPI	NPI	CLI				CMI
Gypsum (Mg/ha)	(g/kg)		(mg/kg)	SOC (%)	(mg/kg)					SMBC	AC	CWC	HWC	SMBC	AC	CWC	HWC
0	16.8a¥	1.99a	149b	0.89b	537b	17.6ba	49.1a	1.01a	1.00a	0.97a	1.00a	1.05a	1.02a	0.98b	1.01a	1.06a	1.03a
1.1	17.6a	2.06a	162ab	0.93ab	547ab	20.7a	55.7ab	1.06a	1.03a	1.03a	1.00a	1.06a	1.05a	1.09a	1.06a	1.12a	1.11a
2.2	17.5a	2.04a	188a	1.08a	562.4a	19.6ab	60.5a	1.05a	1.02a	1.05a	0.99a	1.07a	1.06a	1.10a	1.04a	1.12a	1.11a
Crop rotation
CS	17.2A≠	2.05A	171A	0.99A	532B	20.1A	57.3A	1.03A	1.02A	1.09B	1.00A	1.09A	1.09A	1.12B	1.03A	1.12A	1.12A
SC	17.2A	2.01A	157A	0.91A	553AB	18.9A	52.8A	1.03A	1.00A	1.17AB	1.00A	1.12A	1.14A	1.21AB	1.03A	1.15A	1.17A
SS	17.6A	2.04A	171A	0.99A	561A	18.8A	55.2A	1.06A	1.02A	1.24A	1.01A	1.11A	1.15A	1.31A	1.07A	1.18A	1.22A
Cover crop
Control	17.2x£	2.04x	148y	0.87y	524y	18.5x	58.2x	1.04x	1.02x	1.41x	1.03x	1.14x	1.22x	1.47x	1.07x	1.19x	1.27x
Rye	17.4x	2.03x	185x	1.06x	574x	20.0x	52.0x	1.04x	1.01x	1.44x	1.03x	1.12x	1.22x	1.50x	1.07x	1.16x	1.27x

¥ Means separated by same lower-case letter (a, b, and c) in each column were nonsignificant (at p≤0.05) among gypsum application rates by the LSD test.

≠ Means separated by same upper-case letter (A, B and C) in each column were nonsignificant (at p≤0.05) among crop rotations by the LSD test.

£ Means separated by same lower-case letter (x and y) in each column were nonsignificant (at p≤0.05) between cover crops by the LSD test.

A non-significant increase in SOC pools, TN, and SOC lability was reported by the impact of rye across all sites [29, 43]. This contrasts with expected results that when long-term rotations include a cover crop, there will be pronounced effects leading to an influence on SOC pools. However, we observed limited impacts of rye when used as a winter cover crop due to moderate growth and the high C:N values of cover crop biomass inputs. While rye is a preferred species of winter cover crop across the Midwest, its biomass contribution is highly constrained by the limited growing degree days during the winter and early spring months before planting of corn and soybean crops [28, 29, 43]. We believe that a moderate amount of high C:N ratio (> 80:1) biomass contribution from rye at our sites, when compared to the larger amount of background agronomic crop residues (such as corn), did not contribute to significantly influence SMBC, SOC pools, and lability. This suggests that to take advantage of management practices that include cover crops, the cover crops grown must be adapted to the soil types and climatic zones where they are grown, so that a substantial amount of cover crop biomass is produced. This may be done by using cover crop mixtures and by other practices that promote early and more significant cover crop growth [29].

Interaction of gypsum and cover crops on soil carbon pools and lability

In general, there were few significant interactive effects noted due to treatment combinations. One exception to this was gypsum by cover crop interaction. This was found at most individual sites, but not across all sites or via the meta-analysis. At the individual sites, it seems like the rotation effect is more pronounced at the highest gypsum application rate of 2.2 Mg/ha when compared to the lower gypsum rate or no gypsum application.

Relationship between soil carbon and nitrogen pool (accumulation) indices

When plotted, the SOC and TN pools at each site and across the sites showed a positive and significant linear relationship between them (Fig 2). The exception to this observation was at the Alabama site. The SOC significantly accounted for 88, 89, and 90% of the TN variability in Ohio-Hoytville, Indiana, and Ohio-Piketon, respectively, but at the Alabama site, the R2 value that related SOC and TN was only 0.24. A meta-analysis of data from all sites showed that the SOC significantly explained 89% of the variability in TN with a C:N slope of 10:1.1, which closely conforms to a C:N ration of 10:1 that is accepted for SOM [10, 44].

Fig 2

Relationship between soil total organic carbon (SOC) and total nitrogen (TN) content for a gypsum-amended rainfed transitional no-till soybean-corn rotation with cover crop in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) and averaged across all data.

A similar response between the CPI and NPI values was observed at each site and among the sites (Fig 3). The CPI, a measure of SOC accumulation or depletion over the control, significantly accounted for 77 to 82% of the variability in the NPI, a measure of TN accumulation or depletion over the control, and/or vice-versa in Indiana, Ohio-Piketon, and Ohio-Hoytville. The CPI non-significantly accounted for only 24% of the NPI variability in Alabama. When pooling all the normalized data, 90% of the variability between the CPI and NPI values was accounted for via a strong non-linear relationship. The slope for this relationship had a ratio of 10:0.81, which is slightly different than the standard C:N slope (10:1) in SOM.

Fig 3

Relationship between soil carbon and nitrogen pool indices for a gypsum-amended rainfed transitional no-till soybean-corn rotation with cover crop in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) and averaged across all data.

A significant relationship observed between SOC and TN confirms that the C and N remain strongly coupled in SOM at all sites except marginal soils in Alabama. Although it is reported that C and N remained coupled in SOM, the ratios of C and N may be significantly changed in C-enriched surface soils. The calculated C:N ratios derived from the best-fitted, non-linear relationship between the CPI and NPI values suggest a slightly greater C enrichment in SOM when compared to the N accumulation. This is attributed to a greater amount of surface accumulation of C-enriched unfragmented crop residues (such as CS phases) including rye biomass when used as a cover crop under transitional no-till across the sites. A greater variability observed in the NPI values when correlated with the CPI was related to the more dynamic and labile nature of the total N compared with the SOC. This may also partially account for slightly wider CPI:NPI ratios compared to the conventional C:N ratios observed across the sites.

In accordance with biochemical stoichiometry, the formation of SOM requires a certain amount of N and other nutrients in a fixed ratio with SOC [45]. An increased flow of C-enriched residues (such as corn) in both CS phases than in SS is expected to reduce decomposition of residues and restrict N availability, which may potentially lead to a progressive N limitation in soil [46, 47]. Without new or more N input, the formation of SOM and availability of N is expected to decrease over time under climate change effects. A higher C:N ratio SOM has been observed under CO2 enrichment experiments, which result in slow N mineralization, thus, affecting the release N from SOM to support plant uptake [48]. A higher CPI:NPI observed in our study suggests that a transitional no-till soil ecosystem under climate change effects would need more balanced N fertilization to couple with SOC dynamics for improved soil quality functions.

Relationship between soil carbon accumulation and lability

The CPI, as a measure of SOC accumulation or depletion, showed a significant non-linear inverse relationship with the SOC lability, except for active C (Fig 4b), within and across the sites (Fig 4). The CPI increased with a significant non-linear decrease in SOC lability via SMB and accounted for 41% of its variability across the sites (Fig 4a). The CPI, when correlated with SOC lability associated with cold and hot-water C, accounted for a significant non-linear decrease (by 47% and 42%) of SOC lability (Fig 4c and 4d). In other words, when OC accumulates in soil, it is because its lability has decreased, presumably because the SMBC has processed all of the readily available C into a less labile form.

Fig 4

Relationship between soil carbon pool index and carbon lability indices for a gypsum-amended rainfed transitional no-till soybean-corn rotation with cover crop in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) and averaged across all data.

Higher CPI values were expected to translate into higher SOC accumulation. Moreover, higher values of CPI rather than SOC lability significantly contributed to the higher CMI values with a slightly more non-labile nature of SOC. The high CPI values under both CS phases resulted from surface deposition of a greater amount of C-enriched corn and rye biomass residues with a higher lignin content (~18%), and its consequent effect on the residue and native SOM decomposition [40, 49]. Several studies have reported that surface-deposited organic residues with high C and lignin contents slowed down decomposition and, consequently, accumulated SOC with a higher proportion of non-labile C [3, 50]. Moreover, a longer retention of surface-accumulated crop residues is expected to have greater exposures to environmental variables, especially sunlight, ozone, and UV-radiation, which may lead to an alteration in residue quality and/or palatability for SBMC [51] and thus, slow down residue decomposition with a higher proportion of non-labile to labile C accumulation in SOC [3, 16].

As reported previously [32, 33], our five-year study found no supporting evidence of significantly increased crop yield or farm profitability of either gypsum application or rye cover crop. Given results reported in this manuscript, it is possible that the crop yield and economic performance of the studied practices might improve over a longer timeframe of usage.

Even though our study did not find a direct economic motive for farmer adoption of these practices, the results suggest that there may be positive economic impacts for society more broadly. A reported analysis of soil nutrient content and greenhouse gas emissions for our experimental sites found that cover crops and gypsum usage could result in at least small increases in nutrient availability and decreases in greenhouse gas emissions in certain soil and weather conditions [21, 52]. Increased carbon sequestration offers a value to society broadly through climate change remediation. Hence, policy makers may seek to encourage agricultural management practices that provide such ecosystem services. In addition, carbon markets are emerging that allow high carbon-emitting firms to purchase carbon credits from firms that can demonstrate carbon-reducing production methods or sequestration. Agricultural management practices, such as continuous no-till, crop rotation, cover crops, improved fertility practices, and use of appropriate soil chemical amendments such as gypsum may prove to have sufficient potential to increase SOC sequestration or lessen greenhouse gas emissions to the point where farmers may qualify to participate in carbon offset markets, thereby creating new sources of job and revenue.

Conclusions

Gypsum, when applied at 2.2 Mg gypsum/ha rate, increased labile SOC pools such as SMBC, SMBC:SOC, and active C within and across the sites; however, the CPI and CMI values were not influenced consistently by gypsum application. Crop rotation positively affected SMBC, active C, and SOC lability across all the sites. In contrast, the integration of rye as a winter cover crop exerted a limited impact on the SOC pools. Overall, the SMBC, SMBC:SOC, active C, and CMI via SMBC did show a management-induced impact, thus providing an early indication of any changes in accumulation or depletion in SOC. However, SOC accumulation significantly decreased lability. The interaction of gypsum and cover crops indicates that gypsum applied at the 2.2 Mg/ha rate, coupled with CS rotation, synergistically improves soil biology and SOC dynamics under a rainfed transitional NT system.

(PDF)

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Interactive effects of gypsum, crop rotation, and cover crop on total soil organic carbon (SOC), total nitrogen (TN), microbial biomass (SBM), metabolic quotient (qR), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) at different soil depths under a rainfed transitioning no-till soybean-corn rotation at Alabama site (2012 to 2016).

(DOCX)

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Interactive effects of gypsum, crop rotation, and cover crop on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBM), metabolic quotient (qR), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under a rainfed transitioning no-till soybean-corn rotation at Hoytville site (2012 to 2016).

(DOCX)

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Interactive effects of gypsum, crop rotation, and cover crop on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBM), metabolic quotient (qR), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under a rainfed transitioning no-till soybean-corn rotation at Indiana site (2012 to 2016).

(DOCX)

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Interactive effects of gypsum, crop rotation, and cover crop on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBM), metabolic quotient (qR), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under a rainfed transitioning no-till soybean-corn rotation at Piketon site (2012 to 2016).

(DOCX)

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Interactive effects of gypsum, crop rotation, and cover crop on normalized values of on total soil organic C (SOC), total nitrogen (TN), microbial biomass (SBM), metabolic quotient (qR), active carbon (AC), cold (CWC) and hot (HWC) salt water extractable carbon, carbon pool index (CPI), nitrogen pool index (NPI), carbon lability index (CLI) and carbon management index (CMI) under a rainfed transitioning no-till soybean-corn rotation, averaged across sites (2012 to 2016).

(DOCX)

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1 May 2022

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Besides reviewer's comments, I would emphasize authors should revise manuscript in the following points:

1. Abstract should be written in a simplified language

2. lat-long information should be provided for all study sites, and one US maps pointing all the locations may be added for better understanding.

3. Why Table 1 is pasted in text? In fact all tables should be uploaded separately as per guidelines.

4. As it is multilocational trial, some good photographs of each location's cropping phase, and photographs taken during soil sampling can add values to the MS.

5. Number of references is too high, you can delete some multiple reference; Add some references of 2022 as well.

6. Conclusion should be little generalized (broad implications) apart from summarizing your result; Try to present in bulleted points for more clarity.

7. Please do not put the whole figure caption at P. 24, 25. Only mention Fig. 1 and Fig. 2 suitably.

8. Some track-changed words (at author's end) needs attention during revision.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript “Gypsum, crop rotation and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems” has been reviewed. The MS provides information on SOC and biological properties under different gypsum rate, crop rotation and cover crop.

After going through, the MS is having many fundamental issues which are appended below:

(i) Line 47-49: “SOC is a mixture of diverse and progressively decomposing organic compounds with different complexities and thermodynamic stabilities that greatly affects soil and environmental functions [1-4]”. This sentence is akward. The ‘mixture’ is not best fit.

(ii) Line 49: “It is a composite core indicator” needs modification. It does not sound good.

(iii) Introduction section is large. It needs to concise and simplification (for instance line 129-135 is not needed in Introduction). Also, many sentences are very complex that reduces readability.

(iv) I don’t get the point in MS of adding gypsum in the studied soil. In general, gypsum is added in sodic degraded soil for reclamation. Author must highlight the background for soil status and suggested management. It is also important to clarify whether studied region is having any sodicity problem or not. Only, for carbon sequestration gypsum is added, this statement is not correct. The crop preference and soil geochemical properties drive the management practices in a cropping ecology.

(iv) Line 177-178: Authors mentioned that ‘days under shade at room temperature, processed, and analyzed for chemical and physical analysis (Table 1). However, Table 1 is related to the ‘List of abbreviations and definitions’. The title of table of table needs modification as per text (or vice versa).

(v) Table 1. List of abbreviations and definitions: It is not a format of table (without any column and row): modify accordingly.

(vi) The major fundamental issue is the MS devoid of soil physico-chemical and biological properties of studied soil under ‘Site descriptions’ in MM section. The initial soil properties of different sites are vital for a better idea on studied soil.

(vii) I understand authors must have the yield data of crops. The yield data could be correlated with CPI, NPI for a better clarity on impact of soil properties on yield sustainability.

(vii) Excessive use of abbreviations in MS reduces the readability and clarity in MS.

(viii) In conclusion, it is mentioned that “Gypsum, crop rotation, and cover crop variably affected the depth distribution of SOC pools, TN, 607 and SOC lability without any consistent interactions within- and across the sites”. However, no table is having any mention on depth on which data presented. Only, in MM section it is mentioned 0-15 and 15-30 cm soil sampling depth (line 173). Then, where is depth wise data and how this inference was drawn.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jul 2022

Dear Academic Editor,

Thank you so very much for your proactive comments and suggestions. Please find enclosed a revised copy of our research manuscript entitled “Gypsum, crop rotation and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems” written by Islam, K.R., Dick, W.A., Watts, D.B., Gonzalez, J.M., Fausey, N.R., Flanagan, D.C., Reeder, R.C., VanToai, T., Batte, M.T. for you to evaluate its suitability of publication in PLOS ONE.

This article is one of the outputs of the United Soybean Board funded project # 1520-732-7226; https://urldefense.com/v3/__https://www.unitedsoybean.org/__;!!KGKeukY!28VE9hc_IMjAzCa-S8fUrQhZysThVkv1FBXABtrpCwejhSI5GRcyThExS8QGh8sm9pRPnoNfwNwvx6mRJg$. However, the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

As per your suggestions/comments, the manuscript has been revised to follow PLOS ONE's style and other requirements. Experimental data and details of the statistical analyses were presented as supporting information (S1 File and S1 – S5 Tables).

The abstract of the manuscript was written in a simplified English language expression with a substantial deletion of abbreviations and/or acronyms. All the experimental sites including description, latitude, and longitudes were presented and explained in the map of the United States (Fig 1) and under site description. We have added cover crops, cropping phases, planting, and soil sampling photographs in the U.S. map (Fig 1). Table 1 containing abbreviations and acronyms was removed to avoid confusion.

The number of references cited was reduced to 52 (down from 79) and a few recent references from 2022 were added, including our own works. As per suggestion, the conclusion was revised to be more concise and generalized for easier understanding. A thorough revision of the manuscript was performed with especial attention to track-changed words.

If you have any questions concerning the manuscript, please feel free to communicate with me at your convenience.

___________________________________________

Response to Reviewers' comments:

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No__________________________________

2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No

Response:

A per reviewers’ comments/suggestions, the manuscript was thoroughly revised for better flow, understanding, interpretation, and conclusions presented in simplified English language. All the raw data and their in-depth statistical analysis using SAS and treatment interactions were presented with supporting information (S1 File and S1 Table – S5 Table). Anybody will be able see and use the data, if necessary (with permission). The manuscript was revised and edited by several authors and a professional editor (Bradford Sherman, The Ohio State University Communications).

_________________________________

Response to Specific comments

Reviewer #1: The manuscript “Gypsum, crop rotation and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems” has been reviewed. The MS provides information on SOC and biological properties under different gypsum rate, crop rotation and cover crop. After going through, the MS is having many fundamental issues which are appended below:

(i) Line 47-49: “SOC is a mixture of diverse and progressively decomposing organic compounds with different complexities and thermodynamic stabilities that greatly affects soil and environmental functions [1-4]”. This sentence is akward. The ‘mixture’ is not best fit.

Response: The manuscript was thoroughly revised as per suggestion. The sentence in lines 47-49 was simplified to avoid confusion and for easier understanding.

(ii) Line 49: “It is a composite core indicator” needs modification. It does not sound good.

Response: Simplified as suggested “It is one of the core indicators……….”

(iii) Introduction section is large. It needs to concise and simplification (for instance line 129-135 is not needed in Introduction). Also, many sentences are very complex that reduces readability.

Response: Totally agreed. The introduction has been shortened. The long and complex sentences were simplified to improve flow and readability. As suggested, lines 125 through 135 have been removed.

(iv) I don’t get the point in MS of adding gypsum in the studied soil. In general, gypsum is added in sodic degraded soil for reclamation. Author must highlight the background for soil status and suggested management. It is also important to clarify whether studied region is having any sodicity problem or not. Only, for carbon sequestration gypsum is added, this statement is not correct. The crop preference and soil geochemical properties drive the management practices in a cropping ecology.

Response: Thank you for the comment. For our United Soybean Board-funded project (2012 to 2016), gypsum (flue gas desulfurized gypsum) was applied to improve marginal soil quality to support economic crop productivity, especially corn and soybeans. The soils we have selected to conduct our long-term (still on-going) experiment at all sites were marginal soils (low fertility, compacted, etc.) except Hoytville-Ohio. None of our selected sites have experienced any sodicity or salinity problems. Details of the cropping diversity, cover crops, and soils information at all sites were described in our earlier published papers (Ref. 21, 32, 33, 52). Again, gypsum was not intended to apply for sequestering carbon, but to improve marginal soil quality. A sequence of papers has been published, and more has been submitted/will be submitted for publication with information on soil fertility and chemistry including heavy metals, soil compaction and aggregate stability, greenhouse gas emissions, and inductive and deductive soil quality.

Gypsum application has gained increasing use by farmers around the world, especially in relationship to remediating/reclaiming sodic and degraded soils. While soil carbon sequestration is a topic that has gained much attention in helping alleviate climate change, the information in the manuscript focuses on how gypsum, an agricultural soil amendment, impacts soil carbon fractions and is much needed.

(iv) Line 177-178: Authors mentioned that ‘days under shade at room temperature, processed, and analyzed for chemical and physical analysis (Table 1). However, Table 1 is related to the ‘List of abbreviations and definitions’. The title of table of table needs modification as per text (or vice versa).

Response: Table 1 was removed to avoid confusion of widespread use of abbreviations and/or acronyms. As suggested, a large number of abbreviations were removed and explained accordingly for better flow and readability.

(v) Table 1. List of abbreviations and definitions: It is not a format of table (without any column and row): modify accordingly.

Response: Table 1 has been removed.

(vi) The major fundamental issue is the MS devoid of soil physico-chemical and biological properties of studied soil under ‘Site descriptions’ in MM section. The initial soil properties of different sites are vital for a better idea on studied soil.

Response: Data on selected initial (2012) soil biological, chemical, and physical properties at 0-15 and 15-30 cm depths were briefly described under site description. The information was also listed in our earlier published papers (Ref. 21).

(vii) I understand authors must have the yield data of crops. The yield data could be correlated with CPI, NPI for a better clarity on impact of soil properties on yield sustainability.

Response: Thank you for the comment. Information associated with gypsum, crop rotation, and cover crop impact on crop yields was published in our earlier papers (Ref. 32 and 33). The relationship of CPI and NPI with crop yield will be presented in our upcoming “Inductive and deductive soil quality” papers (manuscript under preparation).

(vii) Excessive use of abbreviations in MS reduces the readability and clarity in MS.

Response: As suggested, the use of abbreviations has been minimized.

(viii) In conclusion, it is mentioned that “Gypsum, crop rotation, and cover crop variably affected the depth distribution of SOC pools, TN, 607 and SOC lability without any consistent interactions within- and across the sites”. However, no table is having any mention on depth on which data presented. Only, in MM section it is mentioned 0-15 and 15-30 cm soil sampling depth (line 173). Then, where is depth wise data and how this inference was drawn.

Response: We did point out that under no-till there will be a stratification effect on soil properties in response to lack of plowing, which was an expected outcome. That is why we did not present the soil depth information in the main tables. However, detailed information on statistical analysis associated with all the main treatments and their interactions, including soil depth, was presented in support information (S1 File and S1 Table – S5 Table).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

1 Aug 2022

3 Sep 2022

Response to Review Comments

Reviewer # 1: (No Response)

Reviewer # 2: This article is a well written manuscript based on an extensive study including field experiments at four different locations. The statistical analyses performed in the study also appears sufficient. However, I could find some issues which the authors should address before considering the article for publication.

Response: The author(s) acknowledged reviewer’s proactive comments and/or contribution to improve the quality of the manuscript. Same credits for the academic editor.

______________________________________________________________________________

Comment: L 31: Write 0-15 and 15-30 cm instead of 0-15 vs. 15-30 cm

Response: Thanks. Corrected accordingly.

Comment: L 46: Nitrogen pool index is mentioned in the keywords, but there is no mention of it in the abstract. At least one sentence in the abstract should mention NPI.

Response: The NPI information was added in the abstract.

Comment: The authors have responded to a query of an earlier reviewer that ‘gypsum was applied to improve marginal soil quality to support economic crop productivity.’ None of the soils in this study was sodic. Then gypsum would only have supplied Ca and SO42-. First of all, if these two were not deficient in the soil (whether sufficient or deficient in the soil was not mentioned in the article), what was the original intent to apply the gypsum. Also, I could not understand how gypsum could improve different labile C pools in soil by only supplying Ca and SO42- if they were not deficient to limit microbial activity. Observing the pH, I don’t think Ca would be deficient in these soils. S may be deficient I don’t know as there is no data on that. If gypsum is not amending any inherent problem like sodicity (which is not the case here as no soil was sodic) or supplying any nutrient which is deficient, then how it has a significant effect on improving soil quality and support economic crop productivity?

Response: While none of the soils were sodic, they were less productive soils, especially Marvyn loamy sand (Shorter, Alabama) and Omulga silt loam (Piketon, Ohio) soils were marginal or degraded soils. While the Marvyn loamy sand is a low water holding capacity, poor aggregate stability, and low SOM content marginal sandy soil, the Omulga silt loam is a compacted fragipan and low SOM content degraded soil.

While most of our studied soil nutrient content data associated with FGD gypsum have already been published (Gonzalez JM, Dick WA, Islam KR, Watts DB, Fausey NR, Flanagan DC, VanToai TT, Batte MT, Reeder RC, Kost D, Shedekar VS. (2022) Gypsum and cereal rye cover crops affect soil chemistry: Trace metals and plant nutrients. Soil Science Society of America Journal. https://wileyonlinelibrary.com/journal/saj2), the exchangeable Ca and S concentration of all the soils were included in the manuscript, as per suggested.

FGD gypsum not only provides Ca and S, but it also provides Mg, K, and micronutrients. The cationic bridging effects of Ca promote flocculation of clay and SOC to form soil macroaggregate stability (>250 µm size aggregates) that lead to accumulated, macroaggregate protected organic C (POC) as particulate organic matter (POM), which is one of the labile C pools. Moreover, gypsum decreases surface crusts formation, acts as an electrolyte to facilitate water infiltration due to its higher solubility than lime, and reduce soluble reactive phosphorus via runoff and tile drainage. All these small but proactive effects of FGD gypsum are expected to improve microbial activity, influence SOC lability and accumulation, and affect crop productivity in conjunction with no-till and cropping diversity with cover crops.

Comment: L 88-90: Mention the type of soil where such results were found

Response: As suggested, a brief description on soil type was added. “Recently, a few studies have reported that gypsum application favors soil biology and enzymatic activity in Hagerstown silt loam (pH 6.2) in Pennsylvania, USA [16] and in alkaline-saline clay soils in northwest China [24]. Gypsum amendments provide several nutrients essential for crop growth in Marvyn loamy sand (marginal soil with poor aggregate stability and low SOM content) in Alabama (Shorter), Blount silt loam in Indiana (Farmland), Hoytville clay in northwestern Ohio, and Omulga silt loam (degraded soil with compaction and low SOM content) in southern Ohio, respectively [21]”.

Comment: L 99: Rewrite the sentence to avoid repetition of similar words

Response: Thanks. Revised accordingly.

Comment: L 189: Write ‘physical properties’ instead of ‘physical analysis’

Response: Thanks. Corrected, as per suggestion.

Comment: L 196: Put the starting parenthesis ‘(‘before ‘SMBC:SOC’)

Response: Edited accordingly.

Comment: L 217-218: Authors have mentioned that “The labile C pool was considered as a portion of the SOC that was comprised of the combined pools of active C, SMBC, and cold and hot-water C.” However, active C, SMBC, and cold- and hot- water C were not extracted sequentially from the same sample; they were extracted from separate samples with different methods. Each of them may a part of the labile C; but they are overlapping themselves. As they are not mutually exclusive, adding them up clearly overestimates the CL, and at the same time underestimates the non-labile C as the later was derived by subtracting CL from total SOC. Hence, the parameters derived from CL are also questionable. The authors should find a way to rectify this issue or give an appropriate rebuttal.

Response: Current statement about the “labile C pool” written in lines 217 - 218 has been corrected. The conceptually defined labile C pools such as active C, SMBC, and cold- and hot- water C were determined by KMnO4 oxidation, extraction with distilled deionized water, or K2SO4 solution using different methods. Separate soil samples were used to measure each C pool. It is not a combined C pool, each C pool was determined separately from SOC. That’s why we have different CL, CLI, and CMI values associated with active C, SMBC, and cold- and hot- water C pools, respectively (Table 1 to 5).

Comment: Table 1, last row: This value of SMBC (1228 mg kg-1) seems too high, might be a typo, please check; If not a typo, then how come so huge difference is not significant?

Response: Thanks. It’s a typo. Corrected accordingly.

Comment: L 280-281: 2.2 kg or Mg ha-1?

Response: Corrected accordingly (2.2 Mg/ha)

Comment: L 317-318: The authors have seen some benefits of 2.2 Mg/ha gypsum application on SMBC, SMBC:SOC, and CMI; but, without the mention of its effect on crop yield, the information remains incomplete. They should at least include the average yield of four years under different treatments.

Response: Thank you for the comment. Detailed information associated with gypsum, crop rotation, and cover crop impact on crop yields was published in our earlier papers (Cited ref # 32 and 33 in the manuscript reference list). As the crop yield data were already published, we have tried to minimize the data overlapping and repetitions in incoming and future papers except soil quality indexing and evaluation.

Cited ref. # 32: Batte MT, Dick WA, Fausey NR, Flanagan DC, Gonzalez JM, Islam R, Reeder R, VanToai T, Watts DB. (2018) Cover crops and gypsum applications: Soybean and corn yield and profitability impacts. American Society of Farm Manager and Rural Appraisers 8:47-71.

Cited ref. # 33: Raut Y, Shedekar V, Islam K, Gonzalez J, Watts D, Dick W, Flanagan D, Fausey N, Batte M, Reeder R, VanToai T. (2020) Soybean yield response to gypsum soil amendment, cover crop and rotation. Agricultural and Environmental Letters. https://doi.org/10.1002/ael2.20020.

Submitted filename: Response to Review Comments-3.docx

Click here for additional data file.

12 Sep 2022

Gypsum, crop rotation and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems

PONE-D-22-07617R2

Dear Dr. Islam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Debarati Bhaduri, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All suggested changes have been suitably addressed.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have tried to address the comments made by me. I have no further comments. The article in its present state may be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

19 Sep 2022

PONE-D-22-07617R2

Gypsum, crop rotation, and cover crop impacts on soil organic carbon and biological dynamics in rainfed transitional no-till corn-soybean systems

Dear Dr. Islam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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