Phosphorus Dynamics in High-Legacy Soils: Acid Phosphatase Activity, Extraction Techniques and Isotherm in Florida Potato Fields

Abstract

In Florida, many agricultural soils contain up to 600 mg/kg of Mehlich-3 extractable phosphorus (P), yet potato growers continue to apply P fertilizers, indicating complex P dynamics that remain underexplored. Previous studies have mainly focused on P fertilizer trials, overlooking crucial factors like phosphatase activity and P sorption isotherms in high-legacy P systems. This study aimed to address this gap by examining acid phosphatase activity (AcPA) and P sorption dynamics in a potato field in northeastern Florida. Utilizing a split-block design, 24 plots were subjected to two P application rates (0 and 49 kg/ha) and three management treatments: a multispecies cover crop (MSCC), MSCC with Telone-C35 (a nematicide), and an untreated control. Significant increases in AcPA were observed during the tuber bulking stage, suggesting that applied P was insufficient for plant needs. P sorption isotherms indicated that the soil had reached maximum P sorption capacity, with applied P primarily fixed through chemical processes. These findings underscore the need for revised P fertilizer strategies in high-legacy P soils and highlight the importance of monitoring AcPA and sorption phases for effective nutrient management.

1. Introduction

Phosphorus (P) is vital for plant growth, forming essential components of biochemical molecules such as adenosine triphosphate (ATP), deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and membrane phospholipids. Common inorganic P fertilizers, including monoammonium phosphate (MAP, 11-55-0), diammonium phosphate (DAP, 18-46-0), and triple superphosphate (TSP, 0-46-0), are widely used due to their water solubility [1]. However, in soils, much of the inorganic phosphate (Pi) exists in cation-bound forms—such as calcium phosphates (Ca-Pi) and magnesium phosphates (Mg-Pi) in high pH soils, and aluminum phosphates (Al-Pi) and iron phosphates (Fe-Pi) in low pH soils. These phosphates are not bioavailable to plants in the native form, and their mobilization depends on the biological activity of rhizosphere microorganisms and root-mediated processes. Similarly, organic phosphate (Po) must be mineralized into plant-available forms through hydrolysis catalyzed by extracellular enzymes like acid phosphatases, which are biosynthesized by plant roots and soil microorganisms in response to P limitation stress [2,3,4].

Inappropriate P management can exacerbate environmental issues. Excessive or poorly timed applications of P fertilizers increase chemical P fixation and potato production costs. The applications also hold a risk of nutrient runoff, which can lead to eutrophication in water bodies, disrupting aquatic ecosystems by promoting algal blooms and depleting oxygen levels critical for aquatic life. Effective management practices, particularly the 5R’s nutrient stewardship [5], including the precise application and timing of P fertilizers, are imperative for mitigating these environmental impacts.

Plants employ a range of adaptive strategies to tolerate phosphorus (P) deficiency, including the increased development of lateral roots and root hairs, as well as the enhanced secretion of root exudates [6,7]. These exudates consist of organic acids, carbohydrates, amino acids, mucilage, chelators, and reductants, which play a critical role in mobilizing insoluble phosphates in the soil. Specifically, organic acids chelate metal cations such as aluminum (Al³⁺) and facilitate the reduction of ferric iron (Fe³⁺), thereby increasing P solubility and availability in the rhizosphere [8]. Under low P conditions, the release of carbon-rich exudates also stimulates microbial activity and accelerates the mineralization of organic P pools, further enhancing P bioavailability to plants [9,10].

Cover crop (CC) mixtures can mobilize insoluble phosphates through the processes and they also diversify P storage pools. This diversification includes organic forms like phytate [11]. Additionally, cover crop (CC) mixtures enhance soil resilience through beneficial microorganisms, such as arbuscular mycorrhizal fungi (AMF), which play a crucial role in crop nutrient uptake, especially in enhancing P influx [12,13].

The P cycle in soil involves various processes such as sorption/desorption and precipitation/dissolution. When excess P is available, soil minerals adsorb phosphate to regulate equilibrium concentrations. Conversely, in P-limiting conditions, soils release or desorb P into the soil solution. The sorption and desorption processes are rapid and pH-dependent [14,15,16,17], while precipitation and dissolution occur more slowly [18]. When there are free metal ions, e.g., Al³⁺ or Fe³⁺ in soil, applied P is readily chemically fixed via precipitation and sorption.

Standard soil phosphorus (P) extractants—such as Mehlich-1, Mehlich-3, Bray-1, Bray-2, Morgan, and Olsen—are designed to target specific soil P pools, which may not accurately reflect the fraction that is bioavailable to plants [19,20]. Thus, evaluating the efficiency of different soil P tests is essential for predicting P bioavailability and estimating P storage capacity in northeastern Florida soils where many agricultural soils are acidic and contain high-legacy P and abundant Al as well as Fe [21,22].

Soil microbes produce acid phosphatase enzymes that are critical under phosphorus-deficient conditions, hydrolyzing organic phosphorus (Po) to release plant-available inorganic phosphate (Pi). Monitoring phosphatase activity at different crop growth stages helps indicate soil organic matter (SOM) mineralization [23,24]. Key soil enzymes in farming systems include urease, phosphatase, dehydrogenase, and β-glucosidase [12,25,26], whose abundance depends on SOM composition and microbial community dynamics [27]. Thus, SOM is a primary driver of soil enzymatic activity and phosphorus recycling [28].

This study aims to evaluate acid phosphatase activity throughout the crop cycle in relation to phosphorus (P) fertilizer application and soil P test results, and to characterize the soil’s P sorption properties. By integrating these findings, the research seeks to inform sustainable P management strategies that reduce environmental risks associated with accumulated legacy soil P, particularly leaching and runoff into the aquifer.

2. Materials and Methods

2.1. In Site Description and Experimental Design

This study began in 2019 at the University of Florida/Institute of Food and Agricultural Sciences (UF/IFAS) Hastings Agricultural Extension Center (HAEC) in northeastern Florida. The soil profile characterization from the Natural Resources Conservation Service (NRCS) Web Soil Survey identified three horizons (A, Bt, and C) composed of sandy marine deposits with 10 diagnostic layers extending to a depth of 230 cm. The soil type is classified as Ellzey (sandy, siliceous, and hyperthermic Arenic Ochraqualf), belonging to the Alfisols order, which is highly leached [29]. The baseline of the soil properties before the study is shown in

Table 1. Initial chemical characteristics of the soil at the experimental site on 19 June 2019.

P Rate (kg ha−1)	Treatments	Soil pH	SOM (%)	P	Ca	Al	Fe	K	Mg	Mn
				kg ha−1
0	Untreated control	5.98 ± 0.2	0.47 ± 0.05	403.79 ± 69.5	789.92 ± 109.4	867.54 ± 231.7	361.47 ± 52.3	49.88 ± 16.8	110.68 ± 23.4	29.14 ± 5.9
	MSCC	5.8 ± 0.1	0.52 ± 0.09	423.7 ± 68.9	763.3 ± 71.5	850.4 ± 162.5	379.4 ± 25.3	60 ± 15.6	101.2 ± 9.9	30 ± 4.7
	MSCC + Telone-C35	5.9 ± 0.1	0.6 ± 0.12	410 ± 16.4	904.8 ± 244.3	881 ± 94	371.3 ± 17.3	58 ± 16.9	114 ± 14.6	31.9 ± 4.1
49	Untreated control	6 ± 0.2	0.51 ± 0.01	403.2 ± 34.9	798.6 ± 68	784.3 ± 57.2	350.5 ± 29.6	51.8 ± 15.3	115.4 ± 6.3	30.5 ± 1.7
	MSCC	5.8 ± 0.1	0.56 ± 0.06	457.6 ± 50.7	876.5 ± 82.8	946.3 ± 91.7	410.5 ± 15.1	62.8 ± 6.6	120.2 ± 16.2	33.9 ± 6.4
	MSCC + Telone-C35	6 ± 0.1	0.52 ± 0.09	426.8 ± 44.4	832.5 ± 44.8	859.7 ± 44.7	364.6 ± 28	58.6 ± 8.9	116.8 ± 13	33.3 ± 2.9

.

The experimental area, approximately 0.3 ha (2749.8 m²), was arranged in a split-block design with six factorial treatments. The main plots consisted of two P₂O₅ application rates: 0 and 112 kg ha⁻¹ (0 and 49 kg ha⁻¹ P). Each main plot was divided into four subplots (80 m²), each with three treatments: 1) untreated control, 2) multispecies cover crop (MSCC), and 3) MSCC plus nematicide (MSCC + Telone-C35). Each treatment was replicated four times (four blocks), totaling 24 subplots. Each subplot had four rows (1.0 m row spacing, 19.8 m long, and 0.2–0.5 m hill height). The untreated control was left fallow during summer, allowing natural weed growth.

An inorganic fertilizer regime of N–P₂O₅–K₂O at 224–112–224 kg ha⁻¹ (equivalent to 224–49–187 kg ha⁻¹ of N–P–K) was implemented. The entire phosphorus input (112 kg ha⁻¹ P₂O₅, equivalent to 49 kg ha⁻¹ P) was applied pre-planting (January 2020 and 2021). Nitrogen and potassium oxide inputs (224 kg ha⁻¹ each) were split into three applications: 50% pre-planting, 25% at emergence, and 25% prior to canopy closure.

In the summers of 2019, 2020, and 2021, multispecies cover crops (MSCCs) were established in all plots except the untreated control. The MSCC mixture included Sunn hemp (Crotalaria juncea L.) and velvet bean (Mucuna pruriens L.), each at 9.1 kg ha⁻¹, and sorghum–sudangrass (Sorghum sudanese (L.) Moench) at 13.6 kg ha⁻¹. Biomass was terminated and incorporated into soil in mid-October, followed by a decomposition period of up to three months (October to January of each year). The three cover crops were mixed to promote root colonization by arbuscular mycorrhizal fungi (AMF) and to increase soil organic matter and organic phosphorus (P) stocks for the subsequent potato crop. Although AMF spore counts and percent root colonization were measured in the summer of 2019, these data are not presented here because the focus of this manuscript is on the potato crop. In the third week of December, Telone-C35 (60% 1,3-dichloropropene and 40% chloropicrin) was applied at 72 L ha⁻¹ to the MSCC + Telone-C35 plots at a depth of 30–35 cm. ‘Atlantic’ potato seed pieces were planted in the third week of January at 20 cm spacing, corresponding to a seed rate of 48,438 seed pieces per hectare.

In MSCC + fumigant plots, Telone-C35 (a mixture of 60% 1,3-dichloropropene and 40% chloropicrin) was applied at 72 L ha⁻¹ to a depth of 30–35 cm in the third week of December. Pre-planting fertilizers were applied in January 2020 and 2021, and ‘Atlantic’ potatoes (Solanum tuberosum L.) were planted at a density of 48,438 seed pieces per hectare.

2.2. Soil Sampling

Soil samples were collected at three growth stages during the potato growing season: pre-planting, tuber bulking, and harvest. During the cover crop growing season, samples were collected at flowering and up to 45 days after harvesting (45-DAH) the cover crops. Soil sample depths were 0–10 cm for pre-planting and harvest, and 0–15 cm for soil chemical properties.

Table 2. Summary of fertilization and soil sampling dates in 2019, 2021, and 2022.

Activities	2019		2020			2021
	Summer	Fall	Spring	Summer	Fall	Spring
MSCC Season
First soil sampling (pre-planting)	19-Jun			15-May
Planting MSCC	22-Jun			23-Jun
Shoot/leave sampling		13-Aug			27-Sep
Root sampling		13-Aug			N/A
Second soil sampling (harvest)		13-Aug			14-Oct
Termination MSCC		22-Sep			15-Oct
Potato Season
Application of Telone-C35		16-Dec			15-Dec
First soil sampling (pre-planting)			13-Jan			11-Jan
Pre-planting fertilization			17-Jan			19-Jan
Planting potato			22-Jan			21-Jan
Emergence fertilization			20-Feb			15-Feb
Layby fertilization			3-Mar			1-Mar
Shoot/leave sampling			N/A			17-Mar
Second soil sampling (flowering)			23-Mar			22-Mar
Harvest potato			20-May			30-Apr
Third soil sampling (harvest)			15-May			10-May

provides a summary of fertilization and soil sampling dates for the 2019, 2021, and 2022 growing seasons. For acid phosphatase activity, soil was sampled from the area under root influence during the tuber bulking stage. Each sample consisted of 30 soil cores (diameter of 2.5 cm) taken from the two center rows of each plot, which were composited and stored in paper bags for nutrient analysis and plastic bags for enzyme analysis. Samples designated for enzyme analysis were maintained at 4 °C in a cooler, while those for nutrient analysis were air-dried at an ambient temperature for at least two weeks before being sieved through a 2 mm mesh. Acid phosphatase activity was measured only in spring 2021 during the potato season, as sample collection and analysis were interrupted in 2020 due to COVID-19 restrictions.

2.3. Soil Extracellular Acid Phosphatase Activity

Acid phosphatase activity was quantified by using a modified fluorometric assay following the procedure of Vrba et al. in 2006 [30]. Briefly, 2 g of air-dried soil was suspended in 10 mL of distilled deionized (DDI) water to form a slurry. The mixture was homogenized using a Tissue Tearor homogenizer (Model 985-370, Biospec Products Inc., Bartlesville, OK, USA) operating at 16,000 rpm for 30 s. A 100 µL aliquot of the resulting suspension was transferred to a 96-well microplate and incubated with 50 µL of modified universal buffer (containing sodium acetate and acetic acid at a pH of 6.0, 5.5, or 5.0) and 100 µL of 500 μM 4-methylumbelliferyl phosphate (MUF-P). The reaction was carried out at 23 °C for 30 min. Fluorescence was measured at an excitation/emission wavelength of 360/460 nm using a BioTek Synergy HT fluorometer (BioTek Instruments, Winooski, VT, USA). Acid phosphatase activity was expressed as the increase in fluorescence over the 30-min incubation period and reported as nmol MUF released per gram of soil per hour (nmol MUF g⁻¹ soil h⁻¹).

2.4. Phosphorus Sorption Parameters

The saturation of soil P binding sites was assessed using the P saturation ratio (PSR) and soil P storage capacity (SPSC) in Mehlich-3 extracts [31]:

$$SoilPSR={\displaystyle \frac{Mehlich3P÷31}{\left(Mehlich3Fe÷56\right)+(Mehlich3Al÷27)}}$$

$$SPSC=\left(ThresholdPSR-SoilPSR\right)\times \left[\left({\displaystyle \frac{Mehlich3Fe}{56}}\right)+\left({\displaystyle \frac{Mehlich3Al}{27}}\right)\times 31\right]$$

The interpretation of the PSR is as follows:

• If PSR < 0.1 and SPSC > 0, the soil is a P sink.
• If PSR = 0.1 and SPSC = 0, the soil is neither a sink nor a source.
• If PSR > 0.1 and SPSC < 0, the soil is a P source.

2.5. Phosphorus Sorption Isotherms

Phosphorus sorption isotherms were determined by equilibrating 1 g of air-dried soil with 10 mL of 0.01 M KCl solution at various P concentrations (0 to 100 mg L⁻¹). Tubes were shaken for 24 h, then centrifuged at 4000 rpm for 20 min using an Eppendorf Centrifuge 5810R (Sigma Aldrich, St. Louis, MO, USA) and the supernatant vacuum filtered through Whatman Grade 41 filter paper with Buchner funnels. Filtrates were colorimetrically analyzed for P within 48 h or frozen for up to 28 days before analysis.

Phosphorus (P) sorption parameters were determined using both the linear adsorption model (Equation (1)) and the Langmuir isotherm model (Equation (2)):

Linear adsorption equation:

$$S=K_d{C}_{24}-S_0 or S=S^{\prime }+S_0$$

(1)

where the following is the case:

S = the total amount of P sorbed, mg kg⁻¹.

S₀ = P originally sorbed on solid phase, mg kg⁻¹.

C₂₄ = P concentration in solution after 24 h, mg L⁻¹.

K_d = slope = the P sorption coefficient, L kg⁻¹.

EPC₀ = intercept = equilibrium P concentration at which net adsorption is zero, mg L⁻¹.

Linearized Langmuir equation:

$${\displaystyle \frac{C_{24}}{S}}={\displaystyle \frac{1}{b{S}_{max}}}+{\displaystyle \frac{C_{24}}{S_{max}}} or S={\displaystyle \frac{\left(S_{max}b{C}_{24}\right)}{1+b{C}_{24}}}$$

(2)

where the following is the case:

S = S′ + S₀, the total amount of P sorbed, mg kg⁻¹.

S′ = P sorbed by solid phase during 24 h, mg kg⁻¹.

S₀ = P originally sorbed on solid phase, mg kg⁻¹.

C₂₄ = P concentration in solution after 24 h, mg L⁻¹.

S_max = P sorption maximum, mg kg⁻¹.

b = a constant related to bonding energy, liter(s) per milligram or (L/mg).

At low concentrations of added P, a plot of S′ versus C₂₄ was used to determine the linear sorption slope (K_d, units = liter(s) per kilogram or L kg⁻¹), S₀, and the equilibrium concentration (EPC₀) at which S′ is zero (added P is neither sorbed nor desorbed). S₀ was then applied to the linearized Langmuir equation above to determine S_max [32].

2.6. Soil Organic Matter (SOM), pH, Mehlich-3 Nutrients, Total P, Morgan P, and Bray-1 P Tests

Soil samples were analyzed for pH and Mehlich-3 nutrients at Waters Agricultural Labs Inc. (Camilla, GA, USA). Soil organic matter was determined by loss on ignition (LOI), and total P was analyzed by dissolving ash in 6 N HCl, followed by the ascorbic acid-molybdenum blue method for phosphate concentration [33,34]. Morgan P [35] and Bray-1 P [19] were extracted and analyzed using standard methods, with phosphate concentration determined colorimetrically.

2.7. Statistical Analyses

Acid phosphatase activity and extractable P concentrations were analyzed using generalized linear mixed models (GLMMs) in SAS 9.4 (SAS Institute, Cary, NC, USA). These variables were treated as continuous, and their distributions were assessed to ensure suitability for analysis under the Gaussian (normal) distribution with an identity link function.

The experimental design followed a split-block structure with double randomization, necessitating the inclusion of both fixed and random effects. Fixed effects included Year, Growth Stage, and P Fertilizer Rate, along with their two-way (Year × Growth Stage) and three-way (Year × Growth Stage × P Rate) interactions. Random effects accounted for variability due to blocks and subplots nested within years, capturing the spatial and temporal heterogeneity of field conditions.

Least-squares means (LSMs) were calculated to estimate treatment effects, and comparisons were made using a significance level of p < 0.01. This stricter threshold was applied to reduce the risk of Type I error associated with the complexity of the design and multiple interactions. Model assumptions, including normality and homogeneity of variance, were evaluated using diagnostic plots and residual analysis.

3. Results

3.1. Acid Phosphatase Activity of Soil Microorganisms

Acid phosphatase activity varied significantly across different potato growth stages (p < 0.0001) but showed no significant response to different P application rates (p = 0.8668) or multispecies cover crop (MSCC) treatments. At pre-planting, AcPA did not correlate with any soil response variables (https://ufdc.ufl.edu/UFE0059679/00001/citation, accessed on 2 July 2025). As the potato plants progressed to the tuber bulking stage, AcPA increased approximately 2.7-fold in both P-fertilized and untreated control plots in Figure 1 in the tuber bulking stage as compared to pre-planting.

This unexpected increase in AcPA during the tuber bulking stage was notable. In 2021, pre-planting measurements indicated Mehlich-3 P (M-3 P) levels of 388 kg ha⁻¹ in untreated control plots and 414 kg ha⁻¹ in P-fertilized plots. In the tuber bulking stage, M-3 P levels had risen by 101 kg ha⁻¹ in untreated control plots and by 80 kg ha⁻¹ in P-fertilized plots. This increase in M-3 P levels suggests the enhanced extractability of P; however, the significant rise in AcPA indicates heightened microbial activity in response to the growing plant’s demand for phosphorus.

To elucidate the dynamics of soil-extractable P,

Table 3. Mean ± SD of Mehlich-3 phosphorus (kg ha⁻¹) by year, crop, phosphorus fertilizer rate, and treatment (June 2019–May 2021).

	Year	2019		2020				2021
	Crop	MSCC		Potato			MSCC	Potato
	Growth Stage	Pre-Plant	Harvest	Pre-Plant	Tuber Bulking	Harvest	Harvest	Pre-Plant	Tuber Bulking	Harvest
P rate (kg ha−1)	Treatment	Mehlich3 P in kg ha−1
0	Untreated control	403.79 ± 69.5	448.06 ± 45	388.66 ± 36.1	423.96 ± 89	432.93 ± 65.1	385.01 ± 50.7	397.9 ± 58.3	409.67 ± 34.8	400.42 ± 54.6
	MSCC	423.68 ± 68.9	433.77 ± 35.8	394.26 ± 48.4	427.88 ± 47.4	439.93 ± 34.8	385.01 ± 26	379.97 ± 45.6	407.43 ± 41.5	418.36 ± 54.3
	MSCC + Telone-C35	409.95 ± 16.4	446.38 ± 20.2	371.28 ± 4	423.4 ± 22.6	405.19 ± 22	366.8 ± 32.3	387.53 ± 16.5	377.45 ± 34	389.22 ± 12.1
49	Untreated control	403.23 ± 34.9	437.97 ± 41	417.8 ± 78	474.12 ± 99.4	421.16 ± 22.4	410.23 ± 53.8	409.95 ± 77.5	505.78 ± 58.8	493.45 ± 59.4
	MSCC	457.59 ± 50.7	430.97 ± 30.8	418.92 ± 62.7	487.29 ± 52.4	490.65 ± 31.8	442.46 ± 25.7	417.8 ± 67	496.82 ± 31.4	506.06 ± 40.8
	MSCC + Telone-C35	426.76 ± 44.4	421.72 ± 42.5	414.71 ± 47.2	514.47 ± 45.9	437.41 ± 45.1	397.06 ± 43.7	414.71 ± 26.7	480.85 ± 35.3	497.38 ± 40.6

reports the mean ± SD of Mehlich-3 phosphorus (kg ha⁻¹) across years, crops, phosphorus fertilizer rates, and treatments from June 2019 to May 2021.

3.2. PSR, SPSC, and Multipoint Isotherm

Soil P saturation ratio (PSR) values greater than 0.1 and soil P storage capacity (SPSC) values less than 0 indicate that the soils in this experiment were likely P-saturated (

Table 4. Soil P saturation indices.

Year	Growth Stage	P Rate (kg ha−1)	PSR	SPSC
2020	Pre-planting	0	0.325	−118.550
		49	0.328	−129.392
	Tuber bulking	0	0.358	−136.575
		49	0.399	−163.892
	Harvest	0	0.383	−140.167
		49	0.409	−151.683
2021	Pre-planting	0	0.325	−120.083
		49	0.350	−131.842
	Tuber bulking	0	0.346	−126.000
		49	0.417	−167.300
	Harvest	0	0.339	−126.367
		49	0.411	−168.242

).

Figure 2 illustrates the three kinetic phases of P in the soil when varying concentrations of P were added to samples from the untreated control in Figure 3A and multispecies cover crop (MSCC) plots in Figure 3B.

Phosphorus (P) sorption in soils typically occurs in three distinct phases. In the first phase, at low solution P concentrations, P is readily sorbed onto available binding sites on soil particles, such as aluminum and iron oxides, clay minerals, and organic matter. In the second phase, as the concentration of P in the soil solution increases, these sorption sites become progressively saturated, leading to a plateau where additional sorption slows or ceases. In the third phase, when the sorption capacity is exceeded, excess P tends to form insoluble precipitates with metal cations (e.g., Al³⁺, Fe³⁺), resulting in chemical precipitation. This sequence reflects the transition from labile to stable P pools, with implications for both fertilizer efficiency and environmental risk.

The isotherm parameters were not significantly affected by the cover crops or the P rates. The S₀ represents the originally sorbed P before planting potatoes in spring 2021. The average baseline P was <10 mg L⁻¹ in all treatment plots (

Table 5. Average sorption parameters for linear adsorption and linearized Langmuir.

Treatment	P Rate (mg kg−1)	Linear Adsorption				Linearized Langmuir
		S0 (mg kg−1)	EPC0 (mg L−1)	Kd (L kg−1)	R2	b (L mg−1)	Smax (mg kg−1)	R2
Untreated control	0	7.47	13.51	0.72	0.88	0.08	171.38	0.74
	49	4.00	3.53	1.18	0.92	0.34	776.88	0.74
MSCC	0	4.98	9.65	0.72	0.92	0.09	399.97	0.71
	49	5.71	11.25	0.91	0.86	0.06	129.17	0.63
MSCC + Telone-C35	0	5.11	6.48	0.83	0.90	0.07	127.74	0.75
	49	6.12	6.59	0.98	0.90	0.10	236.39	0.67

Footnote: there was no statistical difference between treatments.

). The equilibrium phosphorus concentration (EPC₀) is defined as the solution P concentration at which there is no net sorption or desorption [36]. The EPC₀ values were variable and ranged between 3.53 and 13.51 mg L⁻¹. The lower the EPC_0, the better, because if irrigation water has a P load greater than the EPC₀, the soil will act as a sink. Therefore, when P fertilizer is not added to the soil, P is released into the soil. In our study, the K_d ranged between 0.72 and 1.18 L kg⁻¹. Since K_d indicates a soil’s ability to bind P, higher K_d values mean more P is removed from the water column. While the maximum sorption capacity (Sₘₐₓ) is the highest amount of P a soil can sorb, some plots have exceeded their maximum P storage capacity because the Mehlich-3 extractable P was higher than the S_max by 3.6% in the untreated control without P, by 44.3% in MSCC with P, and by 35.3% in MSCC + Telone-C35 without P.

Similarly, the maximum sorption capacity (Sₘₐₓ) was markedly greater in the MSCC-amended soils. Values in MSCC plots ranged from 54.0 to 138.3 mg P kg⁻¹ soil, whereas the untreated control exhibited substantially lower Sₘₐₓ values of 9.8 to 30.8 mg P kg⁻¹. These results suggest that the incorporation of multispecies cover crops enhanced the soil capacity to retain applied P, potentially reducing the risk of P loss via leaching or runoff under high-legacy P conditions.

3.3. Soil Organic Matter, Total P, Mehlich-3, Morgan, and Bray-1 P Tests

3.3.1. Comparison of Soil P Extraction Methods

Comparing soil P extraction methods is crucial for developing the best management practices for P fertilization in high-legacy P cropping systems. During the 2021 potato growing season, Morgan P, Bray-1 P, and Mehlich-3 P extraction tests were significantly influenced by the main effects of P rate and growth stage, as well as by the interaction between P rate and growth stage in

Table 6. Least-squares mean table of different soil P tests during the potato season in 2021.

Main Effects		Morgan P	Bray-1 P	Mehlich-3 P	Total P
		kg ha−1
P Application (PR)
0		69.22 b z	288.52 b	396.44 b	457.19 b
49		99.55 a	347.58 a	469.20 a	497.90 a
p-value		0.0011	0.0027	0.0077	0.0187
SE		±1.70	±14.04	±18.70	±12.93
LSD		7.72	20.51	36.15	27.84
Growth Stage (GS)
Pre-planting		75.60 b	304.76 b	401.31 b	460.40 a
Tuber bulking		87.72 a	326.45 a	446.33 a	496.73 a
At harvest		89.82 a	322.94 a	450.82 a	475.50 a
p-value		0.0120	0.0191	0.0047	0.2432
SE		±2.30	±14.06	±18.74	±16.47
LSD		8.35	14.04	24.55	46.99
Two-Way Interaction		Morgan P	Bray-1 P	Mehlich-3 P	Total P
		kg ha−1
GS	PR
Pre-planting	0	71.03 b	290.72 b	388.47 a	457.02 a
	49	80.17 a	318.81 a	414.15 a	463.78 a
Tuber bulking	0	66.75 b	289.65 b	398.18 b	472.57 b
	49	108.69 a	363.25 a	494.48 a	520.90 a
At harvest	0	69.86 b	285.19 b	402.67 b	441.97 b
	49	109.78 a	360.69 a	498.97 a	509.03 a
p-value		<0.0001	<0.0001	<0.0001	0.1830
SE		±2.87	±14.58	±19.80	±19.46
LSD		7.56	18.85	33.31	42.32

^z Values within columns with a different letter for each soil P test are significantly different based on Fisher’s protected least significant difference (LSD) test with p < 0.01. SE = standard error and LSD = least significant difference.

. In contrast, total soil P differed only between P rates. For all P tests, soil P content was significantly greater in P-fertilized plots than in non-P-fertilized plots before planting, in the tuber bulking stage, and at harvest.

In P-fertilized plots, soil P concentrations were significantly higher in tuber bulking and at harvest compared to pre-planting, reflecting both the applied fertilizer (49 kg ha⁻¹ P) and mobilization of residual soil P. The magnitude of increase in extractable P from pre-planting to tuber bulking varied by the extraction method, underscoring method-specific sensitivities to changes in soil P status. Specifically, the Morgan test indicated an increase of 28.52 kg ha⁻¹, Bray-1 showed 44.44 kg ha⁻¹, and Mehlich-3 detected an increase of 80.33 kg ha⁻¹. These results demonstrate that extraction method selection can substantially influence the interpretation of soil P dynamics in high-legacy P systems.

3.3.2. Soil pH, OM, and Mehlich-3 Extractable Nutrients

During the 2020 and 2021 growing seasons, soil pH changed similarly in both non-P- and P-fertilized plots across each growth stage, indicating that potato growth and fertilization influenced soil pH regardless of P fertilization but was probably related to N and K applications due to both nitrification and cation nutrient uptake. In both years, soil pH decreased by approximately one unit from pre-planting to tuber bulking at both P application rates in

Table 7. Three-way (year × growth stage × P rate) interaction of soil pH and Mehlich-3 P during the 2-year potato.

Three-Way Interaction			Soil pH	M-3 P
Year	Growth Stage	P rate (kg ha−1)		(kg ha−1)	(mg kg−1)
2020	Pre-planting	0	6.2 a z	385 a	171.62 a
		49	6.1 a	417 a	186.08 a
	Tuber bulking	0	5.3 a	425 b	189.62 b
		49	5.3 a	492 a	219.46 a
	At harvest	0	4.9 b	426 a	190.04 a
		49	5.1 a	450 a	200.63 a
2021	Pre-planting	0	5.9 A	388 A	173.29 A
		49	5.9 A	414 A	184.75 A
	Tuber bulking	0	5.1 A	398 B	177.63 B
		49	5.1 A	494 A	220.58 A
	At harvest	0	5.9 A	403 B	179.63 B
		49	5.9 A	499 A	222.58 A
		SE	±0.04	±20.10	±8.97
		LSD	0.09	35	15.43

^z For each year, values within the growth stage and P rate (columns) with a different letter are significantly different based on Fisher’s protected least significant difference (LSD) test with p < 0.01.

. This decline in soil pH was largely independent of P fertilization, except at harvest in 2020, when soil pH in non-P-fertilized plots dropped to 4.9, significantly lower than in other treatments, which ranged from pH 5.1 to 6.2.

Mehlich-3 extractable P was significantly affected by the interaction between the P rate and crop growth stage. In both 2020 and 2021, Mehlich-3 P levels were consistently higher in P-fertilized plots than in unfertilized plots during the tuber bulking stage, and at harvest in 2021. In 2020, pre-planting Mehlich-3 P concentrations were 385 kg ha⁻¹ in unfertilized plots and 417 kg ha⁻¹ in P-fertilized plots. By tuber bulking, levels had increased by 40 kg ha⁻¹ in unfertilized plots and 75 kg ha⁻¹ in fertilized plots. A similar trend was observed in 2021, with Mehlich-3 P increasing by 80 kg ha⁻¹ in fertilized plots and by only 10 kg ha⁻¹ in unfertilized plots. These results reflect the cumulative effect of fertilizer input and the mineralization of legacy soil P, as well as differences in extractable P sensitivity across stages.

Soil organic matter content varied significantly by growth stage. In 2020, SOM was highest at pre-planting and tuber bulking and declined by harvest, likely due to the decomposition of cover crop residues. In 2021, SOM increased from pre-planting to tuber bulking (

Table 8. Two-way (year × growth stage) interaction of soil OM and M-3 nutrients/metal oxides in 2-year potato.

Two-Way Interaction		Soil OM	Ca	Al	Fe	K	Mg	Mn
Year	Growth Stage	%	kg ha−1
2020	Pre-planting	0.51 a z	727 c	892 a	373 b	44 c	74 c	24 c
	Tuber bulking	0.50 a	908 b	862 a	411 a	459 a	101 b	37 a
	At harvest	0.45 b	1021 a	779 b	384 b	196 b	162 a	35 b
2021	Pre-planting	1.02 B	739 B	850 A	386 B	74 B	76 A	27 C
	Tuber bulking	1.07 A	808 A	824 A	415 A	129 A	71 AB	32 A
	At harvest	1.02 B	765 AB	848 A	419 A	103AB	65 B	30 B
SE		±0.03	±29	±43	±17	±12	±4	±0.83
LSD		0.04	22	40	18	35	10	1.7

^z For each year, values within columns with a different letter are significantly different based on Fisher’s protected least significant difference (LSD) test with p < 0.01.

), potentially driven by microbial biomass accumulation, although microbial activity was not directly measured.

Soil calcium (Ca) concentrations increased steadily from pre-planting to harvest in both years. Aluminum (Al) levels remained relatively stable. Soil potassium (K), iron (Fe), and manganese (Mn) concentrations peaked during the tuber bulking stage in both years. The increase in K was attributed to fertilizer inputs, while Fe and Mn trends were likely influenced by changes in soil pH and redox conditions associated with root activity and moisture variation.

4. Discussion

4.1. Phosphatase Activity of Soil Microorganisms and Its Relationship to Soil P Tests

The addition of P fertilizer did not inhibit AcPA activity, indicating that P remained limiting during tuber bulking, even in fertilized plots. This contrasts with findings in forest and grassland ecosystems, where inorganic P suppressed phosphatase activity [3,21]. In this study, P was applied about one week before planting, increasing its contact time with soil minerals; low pH and high Al as well as Fe likely enhanced P sorption, which depends on the equilibrium among soil P pools [37]. While split P applications can reduce sorption and fixation [38], studies show little yield benefit for potatoes in high P soils [39]. Early P is critical for tuber bulking, when photosynthates move to tubers [40], and late applications may not correct P limitation [41]. High tissue P during bulking did not increase yield; greater leaf area index and tissue P accumulation, not just concentration, were associated with higher yields in split-application studies [38]. In unfertilized plots, AcPA and yield correlated positively with leaf P and negatively with leaf Zn, an antagonist to P [42].

The elevated AcPA suggests that standard soil tests (Morgan P, Bray-1 P, and Mehlich-3 P) did not reflect P bioavailability under these conditions, indicating they may be unreliable for guiding P management in northeastern Florida potato production.

4.2. PSR, SPSC, and Multipoint Isotherm

This study showed that soils in Hastings, Florida, had reached their maximum P sorption capacity, consistent with previous research [21,22]. Maximum P sorption ranged from 9.8 to 54 mg kg⁻¹, with higher capacities observed in soils with cover crops. Organic matter and humus enhance surface charges, promoting greater P retention. Once soils reach P saturation, precipitation occurs, rendering P unavailable for plant uptake. These results suggest that the accumulation of high-legacy P soils in Florida is primarily driven by P precipitation, influenced by low soil pH and high Al as well as Fe concentrations (Table 3 and Table 4).

4.3. High AcPA Prompts Split P Applications for Environmental and Economic Sustainability

In northeastern Florida, potato production is challenged by high soil Al (up to 2240 kg ha⁻¹) and Fe (up to 600 kg ha⁻¹) levels and low soil pH (<5) during the growing season [43]. One-time pre-planting P applications often result in substantial fixation by Al and Fe, reducing P bioavailability. Elevated AcPA activity during tuber bulking indicates persistent low P stress despite fertilization.

To address this, optimizing P application strategies during the growing season is critical. Splitting P applications can reduce contact time with Al and Fe, minimizing chemical fixation and enhancing P availability [44]. Field observations in Hastings indicate that some growers have adopted three split applications instead of a single pre-planting dose. During critical growth stages such as tuber bulking, higher P doses may further improve uptake and use efficiency while limiting environmental impacts.

Implementing multiple, smaller P applications supports both economic and environmental sustainability, consistent with precision agriculture principles [45]. Integrating this approach into BMPs for potato and other vegetable crops can improve P management, maintain soil health, and reduce nutrient losses. Ultimately, synchronizing P fertilization with crop nutrient demand is essential for sustainable production, enhancing productivity while preserving natural resources for future generations.

5. Conclusions

This study evaluated acid phosphatase activity (AcPA) across the potato crop cycle in relation to phosphorus (P) fertilization and conventional soil P tests, while also characterizing soil P sorption dynamics. Mixed-species cover cropping (MSCC) and Telone-C35 nematicide application had no significant effect on AcPA or soil P sorption parameters. However, MSCC enhanced the transformation of extractable P into organic forms, increasing microbial accessibility under P-limited conditions.

These results highlight the importance of a site-specific, stepwise approach to P management in high-legacy P soils. Quantifying P sorption capacity through isotherm modeling allows for the identification of soil retention characteristics and informs precise fertilizer applications. Concurrently, monitoring AcPA at critical growth stages, especially during tuber bulking, provides a sensitive indicator of P demand, enabling timely nutrient adjustments to maintain bioavailable P and optimize yield.

Organic P mineralization, mediated by AcPA and microbial activity, is central to this process. In soils approaching P saturation or undergoing precipitation, cover crops facilitate the cycling of P from mineral-bound pools into organic forms, which are subsequently mineralized and rendered plant-available. This biological pathway sustains long-term fertility and productivity while reducing dependence on synthetic inputs.

Importantly, conventional soil P extractants, such as Mehlich-3, may overestimate plant-available P under these conditions, whereas AcPA activity reflects biologically meaningful P availability. Integrating enzyme activity measurements with P sorption analyses provides a robust framework for sustainable P management, improving nutrient use efficiency, supporting crop productivity, and mitigating environmental P losses—contributing to the protection of Florida’s water resources from eutrophication and nutrient pollution.