Soil enzyme activities in heavily manured and waterlogged soil cultivated with ryegrass (Lolium multiflorum)

Abstract Extended waterlogging (WL) conditions can alter soil enzyme activities and their role in maintaining healthy soils. We assessed the effects of soil moisture regimes (field capacity [FC] and WL) and phosphorus (P) rates (0, 15, 30, 45 kg available P ha–1) on (i) soil enzymes and microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial biomass P (MBP); and (ii) dissolved organic carbon (DOC) and total dissolved nitrogen (TDN). The treatments were tested in a 4-month greenhouse experiment using intact soil columns under annual ryegrass (Lolium multiflorum). WL decreased the activity of β-glucosidase and acid phosphomonoesterase but increasedN-acetyl-β-glucosaminidase in soils. These changes were associated with changes in MBC, DOC, MBN, and TDN, but not MBP. Anoxic conditions in WL soil promote the activity of anaerobes and contribute to the reduction of Fe oxyhydroxides and the release of DOC, TDN, and P in the soil solution. The activity of the extracellular enzymes decreased in WL with additions of slurry indicating adequate supply of C, N, and P. Our results also showed that both enzyme activities and microbial biomass were restricted in the upper soil layer with limited downward movement along the soil profile. We can conclude that since these enzymes control the hydrolysis of cellulose, phosphomonoester, and chitin, soil moisture influences the direction and magnitude of C, N, and P in manured and waterlogged soil cultivated with ryegrass.


Introduction
Extreme weather events including heavy rainfall are becoming the norm worldwide in the face of ongoing global changes, posing risks to soil resources.Heavy rainfall can increase the soil moisture content and affect a variety of soil properties and processes (Huang and Hall 2017).Soil microorganisms and extracellular enzymes play a critical role in the formation of soil organic matter and in supplying the soil with nutrients, including carbon (C), nitrogen (N), and phosphorus (P).(Xu et al. 2020).Soil microbial biomass represents important soil nutrient pools with rapid turnover and short mean residence time (Gunina et al. 2017).Soil extracellular enzyme activity is a good indicator of soil physicochemical and biological conditions and microbial status in response to natural disturbances (Utobo and Tewari 2015).Understanding the influence of soil moisture content on changes in activity of soil microorganisms and extracellular enzymes is therefore essential for better management and stewardship of soil resources following heavy rainfall events.
The occurrence of waterlogged or badly drained soils after extreme rainfall events can either directly or indirectly alter extracellular enzymes and soil microorganisms including microbial biomass (Li et al. 2023).Soil microorganisms under extreme conditions such as waterlogging (WL) develop adaptation mechanisms including changes in osmotic activity and reduced oxygen availability (Schimel et al. 2007).Li et al. (2023) found that plant growth-promoting bacteria Pseudomonas was significantly enriched in sugar beet waterlogged rhizospheres compared with the non-waterlogged ones.The authors also found that WL conditions favor the formation of less complex root-microbial network than non-waterlogged ones.Extended excess soil moisture can alter soil enzyme activities more rapidly than other soil variables.They are produced by roots and microorganisms and specifically catalyze the hydrolysis of organic compounds, releasing available nutrients necessary for plants and microorganisms.The extracellular enzyme β-glucosidase is abundant and easily detectable and is involved in the degradation of cellulose (the main component of plant polysaccharides) in soil (Turner et al. 2002).Acid phosphomonoesterase catalyzes the hydrolysis of phosphomonoesters, which account for ∼68%-96% of soil organic P (Turner and Engelbrecht 2011), transforming it into available P (Cabugao et al. 2021).The N-acetyl-βglucosaminidase breaks down chitin, one of the most abundant biopolymers on earth and a primary component of cell walls of common soil fungi (Fuhrmann and Zuberer 2021), converting it into amino sugars which are major sources of mineralizable N (Ekenler and Tabatabai 2004).
In recent studies, Rupngam et al. (2023aRupngam et al. ( , 2023b) ) showed that in heavily manured soils, WL conditions can alter the solubility and mobility of P. The concentration of P in the soil solution as well as microbial activity increased with increasing moisture content (Rupngam et al. 2023a).Long-term anoxic conditions increase the concentration of Fe 2+ and soil pH due to the microbially mediated reduction of metal cations which consumes H + cations.Our objectives were to assess the effects of two soil moisture regimes (field capacity and WL) and P rates (0, 15, 30, and 45 kg available P ha -1 ) on (i) soil enzyme activity (β-glucosidase, N-acetyl-β-glucosaminidase, and acid phosphomonoesterase) and microbial biomass C (MBC), microbial biomass N (MBN), and microbial biomass P (MBP); and (ii) dissolved organic (DOC) and total dissolved N (TDN).We hypothesized that (i) WL conditions decrease soil enzyme activity but increase MB-(C, N, P), DOC, and TDN; and (ii) the application of daily slurry P rates exacerbates the effects of WL conditions.

Site description
The soil used for this study was sampled at the Agassiz Research and Development Center in British Columbia, Canada (49 • 14 34.5 N, 121 • 45 39.3 W).The soil profile belongs to the Monroe series, which is classified as a Gleyed Dystric Brunisol by the Soil Classification Working Group (1998) or a Typic Dystroxerept by U.S. Soil Taxonomy (Soil Survey Staff 2014).The site is covered by grasses, including fescues (Festuca), bluegrass (Poa), dandelion (Taraxacum), clover (Trifolium), and plantain (Plantago).It has been maintained uncultivated and has not been grazed for the past decades.The regional climate is moderate oceanic, with moderately cool and dry summers and warm and rainy winters.The annual rainfall ranges from 1483 to 1689 mm, with the highest levels occurring in November at ∼280-350 mm.The mean daily temperatures in the region range from 3.4 • C in January to 18.8 • C in August, based on a 30-year average recorded at the Agassiz CDA station.

Collection of soil columns
Twenty-four intact soil columns (26.3 cm diameter and 30 cm depth) were collected using PVC pipes (40 cm height, 26.3 cm diameter, and 0.5 cm thickness; see more details in Rupngam et al. (2023b)).Composite soil samples were collected from different pits at 0-15 and 15-30 cm depth, sieved (<2 mm), air-dried, and analyzed for general soil characteristics: soil pH, 4.2; soil textural class, Sandy Loam; and organic matter, 43 g kg -1 (see more details in Rupngam et al. (2023b)).The original grass was cleared from each soil surface by hand, and ∼5.0 g of Italian rye grass (Lolium multiflorum) was seeded into each soil column.

Greenhouse experiment
Eight combinations of treatments including two soil moisture regimes (field capacity [FC] and WL) and four dairy slurry P rates (0, 15, 30, and 45 kg P ha -1 [we assumed that 35% of dairy slurry P is available during the year of the application]) were tested in this study.The treatments were arranged in a randomized complete block design replicated trice for a total of 24 experimental units.The FC was set at 60 ± 10% waterfilled pore space (WFPS), and the corresponding volumetric water content (VWC) was recorded using EM50 datalogger (Decagon Devices Equipment, Pullman, WA, USA) installed at ∼5 cm depth.The average VWC for each treatment was detailed in Rupngam et al. (2023b).The FC treatments were maintained by regular additions of tap water every time the VWC decreased by more than 10%.For the WL treatments, soils were first maintained at FC for 86 days to allow germination, growth, and establishment of the Italian ryegrass.Thereafter, tap water was added to the soil until a layer of water with a height of ∼5 cm above the soil surface was obtained.The WL was then adjusted every time the height of water decreased by ∼2-3 cm and maintained for a total of 124 days.Dairy slurry was collected from a manure pit at the UBC Dairy Education and Research Centre located at Agassiz RDC campus, which is applied on Agassiz RDC lands to grow forage.Dairy slurry was applied before planting ryegrass at rates of 0, 436, 872, and 1308 mL per soil column for 0, 15, 30, and 45 kg P ha -1 , respectively, at the surface of the soil columns and allowed to infiltrate using a 1 L beaker.A supplement of N as urea (46% N) (1626, 1554, 1481, and 1409 mg N for 0, 15, 30, and 45 kg available P ha -1 , respectively) was added to the soil to complement N supply with dairy slurry and meet annual forage N needs according to local recommendation of 300 kg N ha -1 (BCMA 2010).The dairy slurry was analyzed for total N using the Kjeldahl digestion and NH 4 -N using distillation followed by N analysis on the FOSS Kjeltec 2400, and for total P and K using the microwave-assisted acid digestion adapted from EPA 3051 (Peters et al. 2003) followed by ICP analysis.The properties of dairy slurry are reported in Rupngam et al. (2023b).Greenhouse temperature was maintained at 22 ± 1 • C during the day and 18 ± 0.5 • C at night and 16 h day photoperiod (05:00 am-09:00 pm).The natural light was supplemented with 250 μmol PAR•m −2 •s −1 artificial light (photosynthetically active radiation [PAR]; Philips Ceramalux C400S51 high-pressure sodium, P.L Lighting 400 w HID) when outdoor global solar radiation was less than 300 W•m −2 .

Soil sampling and storage
Ten soil cores (0-30 cm depth) were collected from each soil column using an auger (2 cm diameter) at the end of the greenhouse experiment.These soil cores were divided into six layers: 0-5, 5-10, 10-15, 15-20, 20-25, and 25-30 cm.The soil samples were then composited per depth, resulting in 144 composite samples (24 experimental units × 06 soil depths).Each composite sample was divided into two parts: the first was air-dried and sieved (<2 mm) and the second was frozen at -20 • C for further analyses.

Soil enzymes analyses
The assays of soil enzymes are based on microplate fluorescence quantification of the potential activities of βglucosidase (C cycling), N-acetyl-β-glucosaminidase (C and N cycling), and acid phosphomonoesterase (P cycling) enzymes (Deng et al. 2011).The alkaline phosphomonoesterase was not selected for analysis because the initial soil pH is acid (pH 4.2).The reagents used for these assays include (i) modified universal buffer (MUB) (pH 5.5 for the assay of N-acetyl-βglucosaminidase and pH 6.0 for the assay of β-glucosidase and acid phosphomonoesterase) stored at The extraction of enzymes was performed on frozen soils.Briefly, frozen soil was thawed at room temperature for 2 h; then, 1.0 g soil was mixed with 120 mL of deionized water in a beaker for 30 min at 600 rpm on a stir plate.Note that 100 μL of the stirred suspension was pipetted with a multichannel pipette into each well of a black microplate, each containing 50 μL of MUB.Then, 50 μL of MUF substrates was added into each well.The solution in each well was thoroughly mixed by pipetting up and down 10 times.The microplates containing the reaction mixtures were covered with a parafilm and incubated at 37 • C for 1 h in the dark.At the end of the incubation, 50 μL of THAM was added to each well to terminate the enzymatic reaction and increase the signal and detection of fluorescence.The relative signal of fluorescence is stable under the stated conditions for several hours (Deng et al. 2013).Calibration curves were prepared simultaneously with samples using the same procedure, but 50 μL of MUF substrates was replaced by 50 μL of MUF of working standard.The controls were performed using the same procedure, but the substrate was added following the addition of THAM.The autohydrolysis during the incubation period was performed with deionized water in place of soil suspension.The methylumbelliferone (MUF) released by enzymatic hydrolysis of specific substrates was measured immediately upon the termination of the reaction using a fluorescence microplate reader (Synergy HTX multi-mode reader, Biotek) with 365 nm excitation and 450 nm emission.The concentration of MUF was calculated using the calibration curve, and enzyme activities are expressed as nanomoles MUF released g -1 soil h -1 .

Soil microbial biomass analyses
Microbial biomass (MB-) C, N, and P analyses were performed on frozen soils using the chloroform fumigationextraction method (Voroney et al. 2008).For MBC and MBN, the frozen soils were weighted in three sets of 5.0 g.The first set was oven-dried at 105 • C for 48 h to determine moisture content.The second set (non-fumigated) was shaken with 25 mL of 0.5 M K 2 SO 4 in a 50 mL centrifuge tube at 150 rpm for 1 h, centrifuged at 3400 rpm for 8 min, and filtered.For the third set (fumigated), the soils were fumigated with ethanol-free chloroform for 24 h in the dark and then ex-tracted.Fumigated and non-fumigated extracts were stored at -20 • C until analysis using a TOC analyzer (Shimadzu TOC-L CPN Analyzer with Total Nitrogen Unit TNM-L & ASI-L Autosampler).The concentrations of MBC and MBN were calculated using extraction coefficients of 0.35 (K EC ) and 0.5 (K EN ), respectively (Voroney et al. 2008).
To determine MBP, we used four sets of 5.0 g of frozen soils.The first two sets (moisture content determination and fumigation) were treated similarly to MBC and MBN, but the extraction of soils was carried out with 20 mL of 0.5 M NaHCO 3 for 30 min at 150 rpm.For the third and fourth sets, nonfumigated soil samples were placed in glass beakers containing water and soda-lime for 24 h in the dark, then extracted with 20 mL of 0.5 M NaHCO 3 buffered at pH 8.5 and 100 μL deionized water (third set) and 100 μL of a 250 μg P i mL -1 spiking solution (fourth set).The extraction solutions were centrifuged at 3400 rpm for 8 min and filtered.The P concentration in all extracts was determined using the colorimetric blue method (Murphy and Riley 1962), and the MBP concentration was calculated using an extraction coefficient of 0.4 (K EP ) as described by Voroney et al. (2008).

DOC and TDN analyses
The DOC and TDN were determined on air-dried soils.Briefly, 2.5 g of air-dried soil was mixed with 25 mL of deionized water (1:10 w/v soil-to-solution ratio) in 50 mL centrifuged tubes and shaken for 15 min on a reciprocating shaker at a speed of 200 rpm.The soil suspensions were then centrifuged at 4200 rpm for 10 min, and the supernatant was filtered through 0.45 μm filter paper.The filtrates were stored at −20 • C in the dark and analyzed using a TOC analyzer (Shimadzu TOC-L CPN Analyzer with Total Nitrogen Unit TNM-L & ASI-L Autosampler) (Jones and Willett 2006;Liu et al. 2019).

Statistical data analysis
Statistical analyses were performed using the R programming language, including preliminary tests of normality with the Shapiro-Wilk normality test and the homogeneity of variance with Levene's test.The natural logarithm and square root functions were used to convert data with a non-normal distribution, when necessary.Analysis of variance with interaction followed by Tukey's multiple comparison tests (when necessary) were performed at a significance level of 0.05 for all variables.We used principal components analysis (PCA) with the "FactoMineR" and "Factoextra" packages for visualization and identification of correlated variables.In this case, individual and variable biplots were superimposed using the function "fviz_pca_biplot".We performed the correlation test with the "corrplot" package and conducted a significance test using the function "cor.mtest" which generated p-values and confidence intervals for each pair of variables.

Change in soil enzyme activity
The activity of β-glucosidase decreased with increasing soil moisture regime, but the extent was influenced by the dairy slurry rate (p = 0.047) (Table 1).The activity of β-glucosidase Note: ns, not significant at p = 0.05; enzyme activities are expressed as nmol MUF g -1 soil h -1 ; microbial biomass C and N (μg g -1 soil).* = significant at the 5% level; * * = significant at the 1% level; * * * = significant at the 0.1% level; "ns" means not significant at the 5% level; these symbols represent the comparison between two soil moisture regimes at the same slurry rate or soil depth.
was 201 nmol MUF g -1 soil h -1 in WL soil but 415 nmol MUF g -1 soil h -1 in the soil at FC with the application of 45 kg P ha -1 (Fig. 1a).The activity of β-glucosidase was reduced under WL soil at slurry rates 15 and 30 kg P ha -1 , though not significant compared with the soil at FC (Fig. 1a).The activity of β-glucosidase decreased with increasing soil moisture regime, but the extent was influenced by soil depth (p < 0.001) (Table 1).The activity of β-glucosidase at 0-5 cm depth was 677 nmol MUF g -1 soil h -1 in the WL soil and 1075 nmol MUF g -1 soil h -1 in the soil at FC (Fig. 1b).The reduced activity of β-glucosidase in the WL soil was not extended to the lower soil layers (Fig. 1b).

Change in soil microbial biomass C, N, and P
The MBC, MBN, and MBP showed contrasting behaviors with soil moisture content and slurry rate applications (Tables 1 and 2).Among the three microbial biomass indicators, the MBC was significantly affected by the interaction between moisture content and slurry rate (p < 0.001) and varied with soil depth (p < 0.001), while a significant interaction between moisture content and soil depth (p = 0.003) affected the MBN.In contrast, the MBP was not affected by none of the studied factors.The MBC increased under WL compared with FC with the application of 0, 15, and 30 kg P ha -1 of slurry (Fig. 2a).The MBC under WL and FC soils was 841 and 244 μg g -1 with 0 kg P ha -1 of slurry, 1237 and 636 μg g -1 with 15 kg P ha -1 of slurry, and 1359 and 686 μg g -1 with 30 kg P ha -1 of slurry.The MBC also decreased with increasing soil depth from 1134.4 μg g -1 at the 0-5 cm layer to 604.9 μg g -1 at the 25-30 cm layer (Table 1).The MBN was lower under WL (49 μg g -1 ) compare with FC (76 μg g -1 ) in the 0-5 cm soil layer but remained same in the remaining soil layers (Fig. 2b).On average, the MBP was 3041 μg g -1 (Table 2).

Relationship between enzyme activities and chemical soil properties
Principal component analysis demonstrated that the studied soil parameters at the topsoil (0-5 cm depth) were divided into two groups: the variables that were related to (i) field capacity regime and (ii) waterlogged moisture regime (Fig. 3).The three enzymes (β-glucosidase, N-acetyl-βglucosaminidase, and acid phospho-monoesterase), available P (Pw and P M3 ), phosphorus saturation index, concentration of Fe 3+ , and oxidation-reduction potential were related to field capacity moisture regime.The soil pH and concentration of Fe 2+ were related to waterlogged moisture regime.We also identified positive relationships between dry matter yield, P offtake, β-glucosidase, and acid phosphomonoesterase (Fig. 4a).Conversely, Fig. 4b illustrated negative relationships between β-glucosidase and metals such as Mehlich-3 extractable manganese (Mn M3 ), Fe (Fe M3 ), and arsenic (As M3 ), as well as between acid phosphomonoesterase and Mn M3 and Fe M3 .

Changes in soil β-glucosidase and acid phosphomonoesterase, MBC, MBP, and DOC
Extended WL conditions decreased the activity of βglucosidase and acid phosphomonoesterase (Table 1).These results strongly support the hypotheses we formulated.Given that these enzymes are responsible for the hydrolysis of cellulose and phosphomonoester, we can deduce that the direction and magnitude of C and phosphates in soils are influenced by soil moisture.The concentration of MBC (Table 1) Table 2. Activity of N-acetyl-β-glucosaminidase, soil microbial biomass phosphorus (MBP), dissolved organic carbon (DOC), and total dissolved nitrogen (TDN) under annual ryegrass (Lolium multiflorum) as affected by moisture content, dairy slurry rate, and soil depth after 124 days of experiment.

N-Acetyl-β-glucosaminidase
(nmol MUF g -1 soil h -1 ) MBP (μg g -1 soil) DOC (mg kg -1 ) Note: ns, not significant at p = 0.05; SEM, standard errors of the means; means followed by the same letter within a treatment column are not significantly different at the 5% significance level.
and DOC remained high under WL soils, while MBP did not significantly change (Table 2).These results align well with our hypotheses, except for MBP.Microbial biomass P may exhibit a more stable response to changes in environmental conditions or nutrient inputs over a longer time frame.Dairy slurry applications to WL soils can increase the concentration of DOC including glucose and readily available P, thus influencing the release of β-glucosidase and acid phosphomonoesterase produced in rhizosphere and bulk soil by various microorganisms (Liang et al. 2020;Barbosa et al. 2023) that are responsible for the hydrolytic breakdown of cellulose into glucose (Krzyśko-Łupicka et al. 2016) and phosphomonoester into phosphate (Inamdar et al. 2022), that is, available P (Cabugao et al. 2021).In addition, anoxic conditions prevailing in WL soils promote the activity of anaerobes by reducing Fe oxyhydroxides bound to phosphates and organic carbon (OC) (Dunham-Cheatham et al. 2020).The ferric-ferrous ions present in soils are subject to redox reactions, which can compromise the stability of Fe-bound soil organic matter (Adhikari et al. 2016).As a result, available P and OC susceptible to mineralization into labile C are released into soil solution leading to increased concentrations of P and DOC in WL soil.Dairy slurry applications to WL soils increased OC input.Adhikari et al. (2017) found that the reduction of Fe and reductive release of OC was dependent on the C/Fe molar ratio, with high C/Fe ratio enhancing both reduction of Fe and release of OC into soil solution.Finally, it is possible that a feedback mechanism occurs between high concentrations of DOC and phosphates in WL soil with plant roots and microorganisms.One consequence is that the release of the two enzymes β-glucosidase and acid phosphomonoesterase by plant roots and microorganisms is inhibited and therefore the breakdown of cellulose and phosphomonoester.Our results highlighted positive correlations among DOC, soil pH, and Fe 2+ concentration but negative correlations among DOC, oxidation-reduction potential (ORP), and P M3 (Fig. S1).The incomplete mineralization of organic matter under anaerobic conditions (Islam et al. 2021) et al. 2011;Catalán et al. 2017;Wang et al. 2018).These partially degraded compounds can be more soluble and easily dissolved in water, resulting in an increase of DOC (Vink et al. 2010).Our results showed a positive relationship between MBC and DOC (Fig. S1), indicating that the accumulation of DOC under WL soils could stimulate MBC production, as C serves as an energy source for soil microorganisms.WL conditions promote the accumulation of metals cations which in turn have been shown to be related to the inhibition of soil enzyme activity (Liu et al. 2020;Majumder et al. 2022).Dairy slurry additions to soils prone to WL conditions can also exacerbate the accumulation of metals, thus inhibiting soil enzyme activity (Liu et al. 2020;Majumder et al. 2022).Ning et al. (2017) showed that organic amendment applications result into the accumulation of Zn, Cd, and Cr in soils.
Our results showed that the accumulation of Mn M3 , Fe M3 , and As M3 can inhibit the activities of β-glucosidase and acid phosphomonoesterase (Fig. 4b).The formation and proliferation of aerenchyma in adventitious roots under WL conditions are also an indication of the influence of changes in root morphology on the activity of β-glucosidase and acid phosphomonoesterase.It is conceivable that the decreased plant growth under WL diminishes the demand for bioavailable nutrients, thereby decreasing the activity of both enzymes (Fig. 4a).The lack of significant activity of β-glucosidase and acid phosphomonoesterase at lower soil layers is consistent with the distribution pattern of ORP obtained by Rupngam et al. (2023b).
The authors observed that the volume of soil pores was limited at lower depths, thus restricting the excess water to saturate the soil matrix and induce reducing conditions.These limited soil pores at lower depths were due to compaction associated with the use of heavy machinery which causes impairments to the root system.

Changes in N-acetyl-β-glucosaminidase, MBN, and TDN
The activity of N-acetyl-β-glucosaminidase was high following WL conditions.This coincided with high TDN, but not MBN.The results obtained for N-acetyl-β-glucosaminidase and MBN did not align with our hypotheses.However, the observed increase in TDN under WL conditions supports the for- ), dissolved organic carbon (DOC), total dissolved nitrogen (TDN) at 0-5 cm soil depth, and other variables (the analysis methods were published in Rupngam et al. (2023b)) such as dry matter yield (DMY) and P offtake by plant.(b) Correlation between soil enzyme activities and Mehlich-3 extractable metals such as manganese (Mn), iron (Fe), arsenic (As), aluminum (Al), copper (Cu), strontium (Sr), zinc (Zn), and magnesium (Mg) at 0-5 cm depth (the analysis methods were published in Rupngam et al. (2023b)).Significant codes: * * * = p < 0.001, * * = 0.001 < p < 0.01, * = 0.01 < p < 0.05.The colors and their intensity represent the correlation coefficient: blue indicates the positive correlation and red indicates the negative correlation.mulated hypothesis.The enzyme N-acetyl-β-glucosaminidase controls the hydrolysis of chitin in soils.Thus, our results indicate that chitin degradation occurred under anoxic conditions, and this is in line with other studies (Wieczorek et al. 2019;Ali et al. 2021).Under anoxic conditions, chitin mineralization is made by fermenters and Fe reducers through the degradation of carbohydrates derived from chitin-hydrolysis or the scavenging of chitin fermentation products.Chitin is one of the most abundant biopolymers on earth, and its transformation produces amino sugars, the major sources of mineralizable N (Ekenler and Tabatabai 2004).Increased TDN concomitant with increased N-acetyl-β-glucosaminidase under WL conditions is consistent with minor increases of ammonium concentrations obtained in an incubation study by Wieczorek et al. (2019).It is possible that the high activity of N-acetyl-β-glucosaminidase and high TDN obtained under WL soil are directly linked to the increased rate of chitin hydrolysis (Tables 1 and 2).The authors also suggested that N-ammonium consumption through assimilatory pathways and the utilization of nitrate (NO 3 -) as an alternative electron acceptor can deplete the concentration of available N under anoxic conditions.This depletion can induce the activity of N-acetyl-β-glucosaminidase, catalyzing the hydrolysis of chitin, thereby increasing the concentration of more readily available N such as amino sugars.Chitin is a primary component of cell walls of common soil fungi (Fuhrmann and Zuberer 2021), and since fungi make up to 60%-90% of microbial biomass in agricultural soils, fungi are the main source of chitin in such soils (Fernandez and Koide 2012).Anaerobic conditions prevailing under WL soil can result in the death and decay of soil fungi including arbuscular mycorrhizal fungi, thus decreasing the colonization of plant roots by arbuscular mycorrhizal fungi (AMF), the number of entry points per unit of root length colonized (Mendoza et al. 2005), and the diversity of AMF (Deepika and Kothamasi 2015).As the fungal population decreases, chitin becomes a more accessible substrate for N-acetyl-β-glucosaminidase.A decrease in root development and changes in root morphology under WL conditions could potentially result in decreased release of root exudates, leading to a decrease in MBN.The decreased activity of N-acetyl-β-glucosaminidase with 45P compared to 0P aligns with our hypothesis and can be attributed to the accumulation of high concentrations of ammonium (NH 4 + ) and other N forms, including amino sugars from the added dairy slurry.This likely decreases the need of extracellular release of N-acetyl-β-glucosaminidase to break down chitin.In line with β-glucosidase and acid phosphomonoesterase, the activity of N-acetyl-β-glucosaminidase was limited at lower soil depth, and there were no significant effects of WL.This was explained by the limited vertical movement of substrates and limited root development with increasing soil depth.

Practical implications of the findings
The changes in extracellular enzyme activities, microbial biomass, and nutrient availability suggest that WL affects nutrient cycling in the soil.Since the enzymes studied control the hydrolysis of cellulose, phosphomonoester, and chitin, their activity is crucial for nutrient release and availability.The influence of soil moisture on these enzymes indicates that soil moisture content can impact the direction and magnitude of C, N, and P in manured and waterlogged soil.The promotion of anaerobic conditions in waterlogged soil contributes to the reduction of Fe oxyhydroxides and the release of DOC, TDN, available P, and metals into the soil solution, potentially impacting plant growth, overall soil health, and nearby water sources.The decrease in extracellular enzyme activities under WL with slurry additions suggests that the supply of C, N, P, and metals from the slurry may have in-fluenced soil enzyme activity.This has practical implications for managing slurry applications and optimizing nutrient utilization.The restriction of enzyme activities and microbial biomass in the upper soil layer, with limited downward movement, highlights the importance of considering the spatial distribution of soil processes.
The limited soil volume in the soil columns used in our study may alter root-soil interactions, influencing soil enzyme activities.Managing waterlogged conditions in our soil columns could be challenging, therefore influencing water movement in the soil compared with field conditions.The short-term duration of the greenhouse study could limit the ability to assess the long-term effects of manuring and WL on soil enzyme activities.

Conclusions
WL conditions decreased the activity of β-glucosidase and acid phosphomonoesterase but increased N-acetyl-βglucosaminidase in soils.Since these enzymes control the hydrolysis of cellulose, phosphomonoester, and chitin, we can deduce that soil moisture influences the direction and magnitude of C, N, and P in soils.Changes in β-glucosidase and acid phosphomonoesterase under WL soil were related to changes in MBC and DOC, but not MBP.Anoxic conditions in WL soil boost the activity of anaerobes and lead to the reduction of Fe oxyhydroxides that free DOC and phosphates in the soil solution.It is possible that the re-assimilation or re-adsorption of phosphates derived from MBP buffered their changes in the soil solution.High TDN under WL soil concomitant with high N-acetyl-β-glucosaminidase could be ascribed to the increased rate of chitin hydrolysis.

Fig. 1 .
Fig. 1. (a and b) β-Glucosidase as influenced by soil moisture regime, dairy slurry rate, and soil depth.(c and d) Acid phosphomonoesterase as affected by soil moisture regime, dairy slurry rate, and soil depth.Error bars represent standard errors of the means (SEM; n = 144; df = 96).*= significant at the 5% level; * * = significant at the 1% level; * * * = significant at the 0.1% level; "ns" means not significant at the 5% level; these symbols represent the comparison between two soil moisture regimes at the same slurry rate or soil depth.

Fig. 2 .
Fig. 2. (a) Microbial biomass carbon (MBC) as affected by soil moisture regimes (field capacity and waterlogged) and dairy slurry rate.(b) Microbial biomass nitrogen (MBN) as affected by soil moisture regime and soil depth.Error bars represent standard errors of the means (SEM; n = 144; df = 96).* =significant at the 5% level; * * = significant at the 1% level; * * * = significant at the 0.1% level; "ns" means not significant at the 5% level; these symbols represent the comparison between two soil moisture regimes at the same soil depth or slurry rate.

Table 1 .
Results of analysis of variance (ANOVA) of soil enzyme activities, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) under annual ryegrass (Lolium multiflorum) as affected by moisture content, dairy slurry rate, and soil depth after 124 days of experiment.