Long-term application of FYM and fertilizer N improve soil fertility and enzyme activity in 51st wheat cycle under pearl millet-wheat

Our study from an ongoing research experiment initiated in Rabi 1967 at the Research Farm of CCS Haryana Agricultural University, Haryana, India, reports that during the 51st wheat cycle in pearl millet-wheat sequence, adding FYM in both seasons significantly impacted various soil parameters at different wheat growth stages compared to the rabi season. The application of 15 t of FYM ha−1 resulted in a considerable increase in dissolved organic carbon content (9.1–11.2%), available P (9.7–12.1%), and available S (12.6–17.1%), DHA levels by 7.3–22.0%, urease activity (10.1 and 17.0%), β-Glucosidase activity (6.2–8.4%), and APA activity (5.2–10.6%), compared to 10 t FYM ha−1. Application of N120 exhibited a considerable improvement in DHA (11.0–23.2%), β-Glucosidase (9.4–19.2%), urease (13.3–28.3%), and APA (3.3–6.2%) activity compared to control (N0). At stage 3, the box plot revealed that 50% of the available N, P, and S values varied from 223.1 to 287.9 kg ha−1, 53.0 to 98.2 kg ha−1, and 50.0 to 97.6 kg ha−1, respectively. Principal component analysis, with PC1 explaining 94.7% and PC2 explaining 3.15% of the overall variability, and SOC had a polynomial relationship with soil characteristics (R2 = 0.89 to 0.99). Applying FYM15 × N120 treatment during both seasons proved beneficial in sustaining the health of sandy loam soil in North-West India.

(0.36%), Na (0.68%) and Ca (1.25%).Nitrogen was divided into two splits, half applied at the time of sowing and the remaining half after 25-30 days, for both crops under their respective treatments.From the beginning of the trial, only urea has been used as chemical fertilizer in all treatments.Following field preparation, wheat variety WH 1105 was promptly sowed following university recommendations.The wheat crop was harvested manually during the third week of April 2018.The crop was watered using canal water, and an irrigation of approximately 7.5 cm was applied as needed based on a visual assessment of the field.The crops were harvested around 2-5 cm above ground level, and the roots and stubble were then incorporated into the field.

Soil sampling and analysis
Collecting, preparing, and analyzing soil samples involved obtaining two sets of soil samples from each plot at various wheat growth phases, spanning from the fourth week of October 2017 to the third week of April 2018.Soil samples were collected in triplicate from the surface layer (0-15 cm) at five different stages: Stage 1 (at sowing), Stage 2 (tillering), stage 3 (heading), stage 4 (maturity), and stage 5 (after harvesting).A single set of soil samples was air-dried, ground, passed through a 2 mm sieve, and stored in a plastic bag in ambient conditions for physicochemical analysis.Another set of moist soil samples was placed in a freezer at a temperature of 4 °C to be analyzed for enzyme activity.

Physicochemical properties
Sieved soil samples were tested for soil pH using a glass electrode and potentiometric method 29 and electrical conductivity (EC) by adopting the conductometric method 29 .The concentration of soil organic carbon (SOC) was assessed using the wet oxidation method as described by Walkley and Black 30 .Determining dissolved organic carbon (DOC) was conducted using the dichromate acid oxidation method, as described by Ciavatta et al. 31 .The Kjeldahl-distillation method determined the available nitrogen (N), as outlined by Subbaiah and Asija 32 .The available phosphorus (P) in soil samples was assessed by extracting the soil with 0.5 M sodium bicarbonate and measuring P concentration in the extract colorimetrically at 660 nm 33 .Available S was determined by turbidimetric evaluation at 420 nm wavelength 34 .

Enzymes activity
The dehydrogenase activity was assessed by measuring the rate at which tri-phenyl formazan (TPF) was produced from 2,3,5-triphenyl tetrazolium chloride (TTC) as outlined by Casida et al. 35 .The activity of the β-glucosidase enzyme was estimated by determining the amount of p-nitrophenol produced when the soil was incubated with a buffered solution (pH 6.0) containing p-nitrophenyl-β-d-glucoside (PNG) and toluene, following the method described by Eivazi 36 .The urease activity was measured according to the method proposed by Douglas and Bremner 37 .The activity of Alkaline phosphatase (APA) was determined by using the substrate p-nitrophenyl phosphate (PNP) and measuring the absorbance of the yellow colour at 420 nm 38 .

Statistical methods
Statistical analysis was conducted using the software STATISTICA 6.0 by Stat Soft, Inc. (2001).A box plot and principal component analysis (PCA) were conducted to evaluate the variation accounted for by the principal components (PCs) using Origin (Pro) Version 39 .The two most influential principal components, which explained the most significant amount of variability, were visually depicted in a two-dimensional graph.

Results
The long-term effects of different seasons of FYM, levels of FYM, and fertilizer N application on soil fertility and enzyme activity at various stages of wheat growth (stage 1: sowing, stage 2: tillering, stage 3: heading, stage 4: maturity, and stage 5: post-harvest) in sandy loam soil are as follows:

Soil reaction
The soil pH values at stage 1, stage 2, stage 3, stage 4, and stage 5 were 7.64-7.92,7.49-7.80,7.54-7.87,7.49-7.82,and 7.45-7.78,respectively (Table 1).The application methods of FYM had a substantial impact on soil pH, except for stage 5.The use of FYM resulted in the lowest soil pH at all stages of wheat growth.Applying FYM in both seasons resulted in a considerable decrease in soil pH compared to its application in kharif season.During stage 1, the soil pH levels between FYM 15 and FYM 10 treatments and between FYM 10 and FYM 5 treatments were statistically similar.However, beyond this stage, the soil pH values at all FYM levels showed substantial differences.The treatment of 15 t FYM ha −1 resulted in the most substantial decrease in soil pH compared to lesser application rates at all wheat growth stages (Table 1).Applying fertilizer N resulted in a significant decline in soil pH values at all stages evaluated, with the lowest soil pH (7.57) observed at stage 5.All three components generally exhibited a decline in soil pH from stage 1 to stage 5.The box plot revealed that during stage 1, 50% of the soil pH values fell within the range of 7.67-7.82,while during stage 2, the range was somewhat lower at 7.56-7.74.However, the lowest pH values were seen during stage 5, with 50% of the values falling between 7.50 and 7.72 (Fig. 1A).The three components did not significantly interact with soil pH in the current long-term experiment (Data not provided).

Soil electrical conductivity (EC)
The soil EC varied between 0.50 and 0.67 dS m −1 at stage 1, between 0.56 and 0.72 dS m −1 at stage 2, between 0.27 and 0.38 dS m −1 at stage 3, between 0.32 and 0.44 dS m −1 at stage 4, and between 0.28 and 0.39 dS m −1 at stage 5 (Table 1).The different modes, levels of FYM, and chemical N application significantly affected the soil www.nature.com/scientificreports/EC.The soil EC exhibited an opposite pattern compared to the soil pH.In both seasons, the application of FYM resulted in the greatest EC values (ranging from 0.36 to 0.68 dS m −1 ), followed by the single-season application.Compared to stage 1, applying FYM decreased soil EC during rabi, both seasons and kharif season (Table 1).The application rates of FYM had substantially impacted soil EC at all stages.The EC was seen to rise as the FYM rates rose.The soil EC increased during the initial stages of wheat growth until stage 2 and then declined after crop harvest.The box plot revealed that 50% of the soil EC values ranged from 0.59 to 0.72 dS m −1 at stage 2, whereas at stage 5, the range was 0.27-0.36dS m −1 (Fig. 1B).Regardless of the growth stages, the treatment        1).At stage 5, there was a significant interaction between mode × FYM.The maximum soil EC of 0.46 dS m −1 was observed when 15 t FYM ha −1 was applied across both seasons.This was followed by the same dose of FYM supplied in the rabi season (0.41 dS m −1 ) and kharif season (0.30 dS m −1 ) (Fig. 2).A similar pattern was observed when applying 10 t FYM ha −1 , however, all seasons and the rabi season showed no significant difference under the treatment of 5 t FYM ha −1 .Soil EC increased with nitrogen management approaches across all three components and modes of FYM level.However, a decrease in soil EC values was seen at stage 5 compared to stage 1.

Soil organic carbon (SOC)
Throughout 51 cycles of wheat under pearl millet-wheat farming, the SOC levels varied between 0.55 and 1.71% at stage 1, between 0.58 and 2.20% at stage 2, between 0.57 and 2.04% at stage 3, between 0.56 and 1.76% at stage 4, and between 0.55 and 1.73% at stage 5 (Table 1).The different modes of FYM application, quantities of FYM, and application of chemical N in these soils considerably influenced the SOC content.Regardless of the growth stages of wheat, application of FYM in both seasons resulted in a considerable increase in SOC, with the following order of accumulation: both seasons > rabi season > kharif season.The SOC exhibited a rise throughout the initial stages of growth until stage 2, followed by a subsequent drop.However, at stage 5, there was a slight increase in SOC compared to stage 1, except when FYM was added during the kharif season.The box plot indicated that at stage 2, 50% of the results for SOC were within the range of 1.12 to 2.15% (Fig. 1C).
The application of 15 t FYM ha −1 increased SOC content as follows: stage 1: 8.2%, stage 2: 7.3%, stage 3: 7.4%, stage 4: 11.4%, and stage 5: 10.2%, compared to the application of 10 t FYM ha −1 (Table 1).Applying fertilizer N considerably increased the SOC content by 4.9-7.1% at various stages of the wheat growing period.At stage 5, the N 120 treatment resulted in a higher SOC increase than the N 0 treatment (Table 1).The long-term data showed that different combinations of modalities and quantities of FYM substantially impacted the SOC content during all growth stages of wheat (Table 2).The SOC varied between 0.55 and 1.89% at stage 1, 0.58 and 2.36% at stage 2, 0.57 and 2.22% at stage 3, 0.56 and 1.94% at stage 4, and 0.55 and 1.91% at stage 5. Except for rabi × FYM 15 at stage 2, both seasons × FYM 15 showed noticeably increased SOC content at all growth stages compared to either single season × FYM 15 .Regardless of the growth stages, the treatment of both seasons with FYM 15 significantly enhanced the SOC compared to both seasons with FYM 10 , except for stage 2 (Table 2).No significant interactions were observed for SOC in this experiment.However, the specific data on these interactions is not provided.

Soil dissolved organic carbon (DOC)
The soil's DOC levels varied at different growth stages.In stage 1, the range was from 32.5 to 154.3 mg kg −1 .In stage 2, it ranged from 47.5 to 196.5 mg kg −1 .In stage 3, the range was from 81.5 to 312.1 mg kg −1 .In stage 4, it ranged from 50.6 to 228.1 mg kg −1 .Lastly, in stage 5, the range was from 45.2 to 165.8 mg kg −1 .The use of FYM in both seasons significantly increased the DOC content compared to applying it in a single season, regardless of the stages (Table 3).Except for stage 1, the application of FYM during the rabi season yielded much superior results compared to its application during the kharif season.A statistically significant increase in DOC from 5.9 to 20.7% was observed at stage 5 compared to stage 1 of the wheat growing period.The relative abundance of DOC varied depending on the mode of FYM application, with the highest abundance seen in stage 3, followed by stage 4, stage 2, stage 5, and stage 1.At stage 3, the box plot indicated that 50% of the values for DOC fell within the range of 113.9 to 216.2 mg kg −1 (Fig. 1D).The application of 15 t FYM ha −1 resulted in a considerable increase in DOC content at different stages of growth (stage 1: 11.2%, stage 2: 10.4%, stage 3: 10.5%, stage 4: 10.1%, and stage-5: 9.1%), compared to the application of 10 t FYM ha −1 (Table 3).Like SOC, adding N significantly Fig.  3).Although stage 1 had an impact, the levels of modes × level of FYM had a substantial effect on DOC content.In the other growth stages, the application of 15 t FYM ha −1 during both seasons was significantly better.The interaction between FYM and fertilizer N considerably impacted the DOC concentration in soil, similar to the effect observed in modes × amount of FYM (Table 3).Treatment FYM 15 × N 120 resulted in a significantly increased content of DOC compared to treatment FYM 10 × N 120 .However, at stage 2 and stage 3, treatment FYM 15 × N 120 showed no significant difference in DOC content compared to treatment FYM 10 × N 120 .The treatment FYM 15 × N 120 was determined to be more effective in increasing the DOC in the soil compared to other combinations, including the use of FYM and N fertilization (Table 3).The use of fertilizer N in mode × had a considerable impact on soil DOC during stage 3 and stage 4. In both seasons, applying fertilizer N also considerably enhanced DOC levels (Table 3).Ultimately, it was determined that applying FYM 15 × N 120 during both seasons is crucial for increasing the DOC concentration in these soils.Other interactions did not differ significantly for DOC content in soil (Data not given).

Available nutrients in the soil
Available nitrogen (N) The soil's available N content varied from 177.9 to 258.  4).The different seasons of applying FYM had a noticeable effect on the amount of available N, except during stages 3 and 4. The application of FYM in both seasons resulted in the highest soil N levels (240.3-268.4kg ha −1 ) across all growth stages, with the rabi and kharif seasons following closely behind.Applying 15 t FYM ha −1 resulted in a significant rise in available N compared to using 5 t FYM ha −1 at various wheat growth stages.Nevertheless, the application of 15 t FYM ha −1 did not result in a substantial increase in soil available N compared to the application of 10 t FYM ha −1 .The application of fertilizer N 120 resulted in an enhanced availability of N in the soil during various wheat growth stages in the semiarid soils of Haryana (Table 4).The box plot displayed that the median of available N values at stage 3 ranged from 223.1 to 287.9 kg ha −1 (Fig. 1E).Based on the statistical analysis, there were no significant differences in the interactions for the available N in these soils (data not given).

Available phosphorus (P)
The soil's available P levels varied between 26.5 and 101.9, 25.1 and 78.0, 32.1 and 109.3,30.0 and 104.7, and 25.0 and 102.9 kg ha −1 throughout the first, second, third, fourth, and fifth stages of wheat growth, respectively (Table 4).All three parameters have a notable influence on the availability of P in soils.Regardless of the growth phases, applying FYM across both seasons resulted in the highest levels of available P and greatly increased the  4).Nevertheless, the application of FYM during the rabi season was somewhat superior to the application during the kharif season, except for stage 1.Applying FYM during both seasons increased the availability of P compared to its application in the rabi season (Table 4).During the 51st cycle of wheat under pearl millet-wheat cropping, the application of FYM significantly impacted the availability of P in the soil.The order of influence was FYM 15 > FYM 10 > FYM 5 > FYM 0 (Table 4).Compared to the application of 10 t FYM ha −1 , the application of 15 t FYM ha −1 significantly increased the availability of P by 10.4, 12.1, 11.0, 9.7, and 9.9% at stage 1, stage 2, stage 3, stage 4, and stage 5, respectively.The available P levels were lowest at stage 2 and highest at stage 3, with a subsequent drop up to stage 5 (Table 4).The box plot reveals that the lowest level of available P was observed at stage 2, while the highest level was at stage 3. Additionally, at stage 3, 50% of the values for available P varied from 53.0 to 98.2 kg ha −1 (Fig. 1F).Also, applying fertilizer N had a synergistic effect on the availability of P in the soil.It increased available P by 7.5, 9.4, 8.4, 8.9, and 8.4% at stage 1, stage 2, stage 3, stage 4, and stage 5 of wheat growth, respectively (Table 4).The interaction between the level of FYM for soil available P and mode × resulted in a range of 26.5 to 126.3, 25.1 to 94.2, 32.1 to 139.3, 30.0 to 133.1, and 25.0 to 129.9 kg ha −1 at stages 1-5, respectively (Table 2).The interaction between seasons and levels of FYM considerably affected the availability of P in the soil, regardless of the growth phases of wheat.
During stage 2, the use of FYM in the rabi season had a considerably greater benefit compared to the use of FYM in the kharif season.However, the interaction impact of each respective single season with FYM was statistically similar for available P. Throughout all growth phases, in both seasons, the treatment combination of FYM 15 was much better than the other treatments in retaining available P in these sandy loam soils (Table 2).No significant interactions were seen for the availability of P in the soil (data not given).

Available sulphur (S)
The soil's available S content varied from 27.1 to 79.6, 24.2 to 76.0, 31.6 to 105.8, 30.4 to 95.4, and 28.5 to 83.8 kg ha −1 throughout Stage 1, stage 2, stage 3, stage 4, and stage 5 of wheat growth, respectively (Table 4).All three elements of nutrient management impacted the availability of S in the soil.Irrespective of the phases, applying FYM during both seasons considerably increased the availability of S content in the soil by 20.0 to 27.9% compared to the rabi season.The order of effectiveness was found to be: both seasons > rabi season > kharif season (Table 4).The application of FYM had a considerable impact on the soil's available S content, with the highest effect observed in the FYM 15 treatment, followed by FYM 10 , FYM 5 , and FYM 0 (Table 4).Applying 10 t FYM ha −1 resulted in a considerable increase in available S at stage 1, Stage 2, stage 3, stage 4, and Stage 5.However, when a higher rate of 15 t FYM ha −1 was applied, the rise in available S was even more significant, with percentages of 14.4, 17.1, 12.6, 16.8, and 15.1% at each corresponding stage.The application of fertilizer N in wheat cultivation resulted in a considerable increase (8.2 to 9.7%) in the amount of available S in the soil compared to plots not treated with N (Table 4).The box plot indicated that at stage 3, the range of available S values spanned from 50.0 to 97.6, with 50% of the data falling within this range (Fig. 1G).The interaction between mode × level of FYM had a significant impact.The available S ranged from 27.1 to 99.2, 24.2 to 93.3, 31.6 to 128.1, 30.4 to 116.0, and 28.5 to 106.7 kg ha −1 during stages 1-5, respectively (Table 2).Throughout all stages of wheat growth, applying FYM resulted in the highest levels of available S in both the rabi and kharif seasons.Regardless of the growth phases, both seasons × FYM 15 treatment combination exhibited superior performance compared to the other treatments in enhancing soil S availability (Table 2).The remaining interaction for the accessible S in the soil was not statistically significant (data not given).5).Except for stage 1, the application of FYM during both seasons had a considerable positive impact on DHA compared to its application in either the rabi season or the kharif season.The order of effectiveness of the application modes was as follows: both seasons > rabi season > kharif season.Applying FYM during the rabi season resulted in a notable rise in DHA levels compared to the kharif season at stage 2 and stage 4 (Table 5).The application of FYM positively affected DHA during the entire wheat growth period.Treatment FYM 15 showed a considerable improvement in enhancing the production of DHA at stage 2, stage 3, and stage 4, whereas it was statistically similar to treatment FYM 10 in all other aspects.Nevertheless, the utilization of 15 t FYM resulted in a notable rise in DHA levels.Specifically, there was a substantial increase of 19.8, 11.7, 22.0, 7.8, and 7.3% at stage 1, stage 2, stage 3, stage 4, and stage 5, respectively, compared to the FYM 10 treatment (Table 5).Fertilizer N application was found to be crucial and significantly improved the development of wheat at various stages, regardless of growth stage.The value of DHA was more than 100% at stage 5 compared to its corresponding value at stage 1, among various treatment combinations.Thus, each of the three variables significantly enhanced and sustained DHA levels over wheat's growth phase (Table 5).The box plot indicated that the level of DHA was highest in stage 3, followed by stage 4, stage 2, stage 5, and stage 1 (Fig. 1H).The interaction between modes and level of FYM significantly affected DHA at stages 2-4.The range of DHA values were 64.5-211.0,97.6-279.0,and 73.8-249.2µg TPF g −1 24 h −1 , respectively (Table S1).The application of FYM during the rabi season had a statistically significant impact on DHA at stage 2 and stage 3.However, at stage 4, the combination of rabi × FYM 10-15 was found to be superior in enhancing DHA compared to the comparable combination under kharif × FYM 10-15 (Table S1).The impact of other interactions on DHA was determined to be statistically insignificant in these sandy loam soils (Data not provided).

β-Glucosidase activity
The soil's β-Glucosidase activity ranged from 57.7 to 119.5, 83.8 to 209.0, 106.3 to 318.7, 169.2 to 449.0, and 146.1 to 414.0 µg PNP g −1 h −1 during the stage 1, stage 2 stage 3, stage 4, and stage 5 of wheat growth, respectively (Table 5).β-Glucosidase activity during the wheat growing phase was significantly affected by the seasons, levels of FYM, and chemical N treatment, regardless of the stages.Irrespective of the phases, both seasons of FYM application exhibited increased β-Glucosidase activity compared to the rabi season.The activity levels were as follows: stage-1: 12.4, stage-2: 11.7, stage-3: 11.4, stage-4: 7.2, and stage-5: 10.9% (Table 5).Applying 10 t, FYM ha −1 resulted in a β-Glucosidase activity equal to the higher rate only during stage 1.However, from stage 2 to stage 5, the application of 15 t FYM ha −1 was much more effective.The maximum β-Glucosidase activity was seen during stage 4 of the wheat growing period, regardless of FYM levels (Table 5).Fertilization with N greatly enhanced β-Glucosidase activity throughout all phases of wheat growth.Treatment N 120 significantly increased β-Glucosidase activity at stages 1-5, with percentage increases of 14.0, 19.2, 15.4, 9.8, and 9.4% compared to treatment N 0 (Table 5).The box plot revealed that the β-Glucosidase activity was most pronounced during stage 4 of wheat growth, in comparison to the other growth stages (Fig. 1I).The interaction effect of modes × FYM on β-Glucosidase activity varied between 83.8 and 232.6, 169.2 and 485.7, and 146.1 and 451.9 (µg PNP g −1 h −1 ) at stage 2, stage 4, and stage 5, respectively (Table S1).Regardless of the levels of FYM, both seasons × FYM exhibited much better performance than either single season.Additionally, rabi × FYM outperformed kharif season × FYM specifically for stage 4 to stage 5.The impact of both seasons × FYM 15 on β-Glucosidase activity was substantially higher than all other combinations.The relationship between the levels of fertilizer N and the activity of β-Glucosidase was found to be considerable, similar to the relationship shown in modes of FYM (Fig. 3).The combination of FYM 15 × N 120 had a significant effect compared to other combinations at stage 2 (223.0 µg PNP g −1 h −1 ), stage 4 (469.6 µg PNP g −1 h −1 ), and stage 5 (427.5 µg PNP g −1 h −1 ) during the wheat growing period (Fig. 3).However, there were no significant differences seen in the interactions during the other growth phases of wheat (Data not provided).

Urease activity
The soil's urease activity varied between 15.1 to 27.7, 13.0 to 21.9, 31.8 to 87.9, 22.8 to 65.5, and 15.2 to 40.0 µg NH 4 + -N g −1 h −1 throughout the stage 1, stage 2 stage 3, stage 4, and stage 5 of wheat growth, respectively (Table 5).Compared to each season, applying FYM considerably enhanced urease activity in all wheat growth stages, except for the kharif season, which performed similarly to both seasons.At stage 1, the urease activity was 22.1 µg NH 4 + -N g −1 h −1 for the kharif season and 24.0 µg NH 4 + -N g −1 h −1 for both seasons.The administration of FYM improved urease activity by 12.1, 12.6, 7.8, 21.9, and 17.5% at stages 1 to 5, respectively, across both seasons compared to the rabi season (Table 5).Regardless of the growth phases, the positive effect of FYM levels was seen, and urease activity increased as the level of FYM grew (Table 5).At stages 1-5, treatment FYM 15 increased urease activity by 13.5, 10.1, 13.0, 16.8, and 17.0% compared to the control treatment FYM 10 .However, there was no significant difference between FYM 10 and FYM 15 at stage 2 and stage 5. Plots treated with fertilizer N showed a considerable increase in urease activity compared to control circumstances (N 0 ).The rise in urease activity was 13.3, 15.5, 18.0, 28.3, and 26.9% at stages 1 to 5, respectively, compared to the control plots (N 0 ) (Table 5).During the growing period of wheat, the order of urease activity among the phases of wheat growth was stage 3 > stage 4 > stage 5 > stage 1 > stage 2. A box plot displayed that 50% of the values for urease activity varied from 50.3 to 82.1 µg NH 4 + -N g −1 h −1 at stage 3 (Fig. 1J).The interaction between mode × amount of FYM was found to be significant at stage 3.The range of NH 4 + -N release was seen to be from 31.8 to 96.4 µg NH 4 + -N g −1 h −1 (Fig. 4).No substantial impact on urease activity was seen in any other interactions (Data not provided)., respectively (Fig. 5).The application methods of FYM had a substantial impact on the APA similar to the available P in the soil.During various stages, the APA showed a rise of 4.4 to 10.6% when both seasons FYM were applied during the rabi season.However, it declined at stage 2 and then consistently increased up to stage 5 (Fig. 5).At stage 3 of wheat growth, the APA reached its peak.A box plot analysis revealed that 50% of the values ranged from 590.8 to 848.1 µg PNP g −1 h −1 at this specific growth stage (Fig. 1K).Irrespective of the phases, every level of FYM positively impacted APA.The application of 15 t FYM ha −1 was found to be more effective in enhancing APA than lower dosages.The application of fertilizer N had a significant impact on APA at various phases of the wheat crop (Fig. 5).Out of all the interactive effects, only the combination of mode × amount of FYM positively impacted APA at all stages.The APA values ranged from 228.5 to 499.6, 432.7 to 843.0, 459.4 to 924.4,372.8 to 715.5, and 278.1 to 601.5 µg PNP g −1 h −1 at stages 1 to 5, respectively (Fig. 6).A notable influence of various levels of FYM on APA was observed in the following order: both seasons and level of FYM had the most significant impact, followed by the rabi season and level of FYM, and finally the kharif season and level of FYM.However, the trend for a single season was inverted for stage 1 (Fig. 6).There was no significant effect on APA from any other interactions independent of the growth phases of wheat (data not provided).

Discussions Soil reaction
Soil pH is a crucial soil characteristic that provides a comprehensive understanding of the environment for plant growth, including nutrient availability, behavior of additional fertilizers, salinity/sodicity levels, soil aeration, soil mineral composition, and the prevailing meteorological conditions of the region 40 .Applying FYM for 51 years in the pearl millet-wheat cropping cycle resulted in a drop in soil pH relative to its initial value of 8.2.The use of FYM resulted in the lowest soil pH at all stages of wheat over both seasons, as shown in Table 1.The application of 15 t FYM ha −1 resulted in the most considerable decrease in soil pH compared to lesser FYM rates, regardless of wheat growth phases.Multiple studies have documented a reduction in soil pH following organic manures 41 .Applying fertilizer N resulted in a significant decrease in soil pH values at all stages of wheat growth.The lowest soil pH (7.57) was observed at stage 5.The trend indicated that soil pH was first at its greatest during stage 1 and then declined from stage 3 to stage 5 (Fig. 1A).Regardless of the types and amounts of organic fertilizer used, soil pH dropped during the growth phases of the wheat crop compared to the time of sowing.This decline could be attributed to the oxidation of ammonium to nitrate that occurs during the growth of crops.Humic compounds include many functional groups, such as carboxylic acids and phenolics, within their organic matrix.The drop in soil pH can be attributed to specific properties such as pH buffering, regulation of equilibrium in ion exchange, and oxidation-reduction processes 42 .The prolonged utilization of chemical fertilizers, specifically urea, as an N source has led to soil acidification 43 .This is because these fertilizers provide N in the form of NH 4 + , which, when oxidized, releases H + ions into the soil.Over 33 years, the continuous use of FYM and mineral fertilizer in rain-fed soybean-wheat cropping systems resulted in a considerable drop in soil pH 44 .Meena et al. 45 found that applying FYM and sewage sludge to sandy loam soil under irrigated circumstances in a pearl millet-wheat cropping system decreased soil pH.Sharma and Subehia 46 observed a positive effect on soil pH in the rice-wheat system after 20 years of using organic and inorganic sources.

Electrical conductivity (EC)
The soil EC was significantly affected by different modes, levels of FYM, and fertilizer N application.Regardless of the growth stages, both seasons and FYM 15 treatment exhibited the maximum soil EC independently.The concentration of soluble salts in water extract (soil EC) is directly proportional to the amount of organic manure applied 47,48 .In comparison to stage 1, the application of FYM resulted in a decrease in soil EC by 40.4%, 41.3%, and 51.7% at stage 5, with rabi, both seasons and Kharif season, respectively (Table 1).The breakdown of organic manures results in the release of organic acids/compounds that react with sparingly soluble salts already present in the soil.This process either converts them into soluble salts or increases the solubility of salts 49 .Relative to stage 1, soil EC dropped 36.0% to 50.0% at stage 5 when varied amounts of FYM were applied.Except for stage 1, the application of fertilizer N significantly increased the soil EC.However, the EC dropped as the growth period progressed.Additionally, the use of chemical N lowered the EC by 42.6% at stage 5 compared to stage 1 (Table 1).The soil EC showed periodic fluctuations, with the maximum recorded at stage 2 and a considerable drop observed afterwards (Fig. 1B).The rise in EC may be attributed to the introduction of soluble salts through the addition of FYM and the presence of soluble salts in N fertilizer.The irregular pattern observed may be attributed to the mineralization of FYM during the initial phases of crop growth, which led to an increase in soil EC.Additionally, the re-adsorption of cations and anions on the soil surface during later crop growth stages could have contributed to this irregular trend 50 .Applying FYM and sewage sludge at higher rates resulted in a considerable increase in soil EC in sandy loam soil's irrigated pearl millet-mustard cropping system 45 .According to Escobar and Hue 50 , introducing organic materials into andisols increased soil EC; conversely, EC in oxisols and ultisols initially increased during the incubation period but declined subsequently.Soil EC increased with nitrogen management strategies.However, a decrease in soil EC was observed at stage 5 compared to stage 1 of wheat.The soil EC values at stage 5 were significantly lower than the safe limit (0.80 dS m −1 ) for crop development in sandy loam soils of Haryana.

Soil organic carbon (SOC)
The SOC is an essential characteristic of soil health which acts as a soil conditioner, provides nutrients, serves as a substrate for microbial activity, protects the environment, and maintains crop yield 22 .The incorporation of organic materials may have increased microbial activity in the soil, leading to the formation of water-stable macro-aggregates.These aggregates are primarily held together by fungal hyphae, fibrous roots, and polysaccharides, as described by Kumari et al. 1 .The FYM application in both seasons showed greater effectiveness than either single season, resulting in a 6.5 to 11.5% improvement in SOC compared to rabi season (Table 1).The application of 15 t FYM ha −1 resulted in considerably higher SOC content than 10 t FYM ha −1 for the entire wheat growth period.Applying fertilizer N also resulted in a considerable increase in SOC content, ranging from 4.9 to 7.1% at various wheat growth stages.Specifically, at stage 5, treatment N 120 was found to raise SOC by 6.9% compared to treatment N 0 , as shown in Table 1.The reason for this could be the increased crop productivity resulting from applying nitrogen fertilizers, which leads to a higher deposit of SOC 51 .The SOC increased until stage 2 and then consistently reduced.However, it was somewhat higher at stage 5 compared to stage 1 (Fig. 1C).The rise in SOC content from sowing to tillering could be attributed to the decomposition of carbon compounds facilitated by FYM application.The decline in SOC content from the growth stage to maturity could be attributed to the enhanced consumption of carbonaceous compounds by a larger population of microorganisms and higher organic matter losses as carbon dioxide (CO 2 ).The pattern of OC content showed an initial increase followed by a decline toward maturity, which aligns with the observations made by Islam and Borthakur 24 .The long-term data showed that different amounts of FYM substantially impacted SOC content at all phases of wheat growth (Table 2).Overall, both seasons × FYM 15 showed significantly greater ability to sustain the greatest level of SOC over the wheat growing period as shown in Table 2. Applying organic manures consistently, either alone or in combination with fertilizers, led to higher SOC levels than inorganic fertilizers alone, as demonstrated by studies conducted by Yaduvanshi et al. 52 .In a study conducted by Zhang et al. 53 , it was found that continuous fertilizing over 26 years increased SOC, ranging from 19.2 to 58.2% compared to the original value.The highest increase was observed in plots treated with NPK fertilizer and organic manure.The use of organic manures in combination with fertilizers has consistently resulted in the most significant OC content, as documented by several researchers 27 .Lemanowicz et al. 54 found that the highest SOC content, measuring 8.22 g kg −1 , was obtained while applying 60 Mg ha −1 of FYM.However, increasing the FYM application rate led to a 4% decrease in SOC content.The study also found a polynomial correlation between SOC and soil characteristics with an R 2 value ranging from 0.89 to 0.99 (Fig. 7).

Dissolved organic carbon (DOC)
Chemical extraction techniques have traditionally been used to measure labile carbon pools, which are considered early and sensitive indicators of management-induced changes in the quality and composition of SOM 11,55 .Applying FYM in both seasons resulted in a considerable increase in DOC content compared to applying it in a single season, regardless of the stages (Table 3).There was a significant increase in DOC from 5.9 to 20.7% at stage 5 compared to stage 1.The relative abundance of DOC was measured during the growing period of wheat at stage 3, as shown in Fig. 1D.The inclusion of FYM may have enhanced microbial growth due to an ample supply of nutrients, hence expediting the decomposition of organic matter and elevating the concentration of DOC 56 .The application of 15 t FYM ha −1 resulted in a considerable increase in DOC content compared to the application of 10 t FYM ha −1 (Table 3).Like SOC, adding nitrogen fertilization considerably raised the DOC content from 28.0 to 43.9% during different growth stages of wheat.Except for stage 1, the various modes and levels of FYM significantly affected DOC content.Applying 15 t FYM ha −1 in both seasons was the most effective combination for raising the DOC content in semi-arid soils during subsequent growth stages.The combination of treatment FYM and chemical N substantially interacted with DOC content in the soil, as shown in Table 3. Applying FYM 15 xN 120 during both seasons was crucial for increasing the DOC content in these soils.Applying inorganic fertilizers and organic manures has a considerable impact on the concentration of DOC in soil 55 .The treatment of the combination of NPK and FYM at a rate of 10 t ha −1 year −1 to a sandy loam soil under a maizewheat cropping system for 34 years resulted in a considerable increase in DOC compared to soils treated with only N, NP, or NPK 57 .There has been a notable rise in DOC levels when N is applied compared to the control.Using compost alone or in combination with N for 22 years significantly raised DOC levels in soils under a maize-wheat cropping system 58 .Banger et al. 59 found that applying inorganic fertilizers and organic manure to sandy loam soil for 16 years caused significant changes in DOC compared to SOC in a rice-cowpea farming system.Escobar and Hue 50 found that the concentration of soluble organic carbon reduced exponentially with time due to soil microorganisms' conversion of organic carbon to carbon dioxide.Bastida et al. 60 also noted a rapid decline in DOC initially attributed to enhanced mineralization.The further drop in DOC at later stages may be attributed to the oxidation of soluble carbon as CO 2 and its crop consumption.The study also found a polynomial correlation between SOC and DOC concentration, with an R 2 value of 0.89 (Fig. 7).

Nutrient availability in soil
Available N The numerical value of available N increased till stage 3. Statistically, applying FYM during both seasons was more effective than applying it during a single season at stage 5 of wheat.However, the different FYM application methods did not maintain N availability in the soil at Stage 1 and Stage 5 (Table 4).This phenomenon could be explained by the leaching and volatilization loss of N under prevailing semi-arid circumstances in addition to the plant's usage 61 .Adding organic manure has improved the soil's physical, chemical, and microbiological characteristics, increasing the availability of N in soil 62 .The application of 15 t FYM ha −1 resulted in a substantial increase of 6.1 to 7.2% in N availability compared to the application of 5 t FYM ha −1 .However, similar to SOC and DOC content, the application of 15 t FYM ha −1 did not result in a substantial increase in soil available N compared to the treatment of 10 t FYM ha −1 .Applying chemical fertilization (N 120 ) increased N availability in soil from 9.1 to 11.2% during various growth stages of wheat in the soils of semiarid regions (Table 4).Hassan et al. 63 found the highest levels of available N and P with the application of 100% of N through FYM followed by the treatment using 75% N through FYM and 25% from urea.Several research studies have found that using INM resulted in a considerable increase in available N compared to using organic manure or fertilizers alone 64 .Prakash et al. 12 reported that the use of fertilizers, either alone or in combination with organic materials, yielded significantly better results compared to using organic manures alone.The frequent application of organic manure increased N mineralization, and the concentration of NH 4 -N released was higher with higher application rates of manures 14 .Basak and Biswas 65 reported that applying organic manures with fertilizers showed the highest levels of mineral N, compared to using manure alone.Similar to DOC, the maximum amount of available N was observed at stage 3, followed by stage 2, stage 4, stage 5, and stage 1 (Fig. 1E).The availability of N from inorganic sources is fast but only for a limited period.On the other hand, when organic manure is applied, the availability of N is longer due to the production of complex polyphenolic compounds with high molecular weight.The early-stage increase in available N content is primarily caused by the quick hydrolysis of urea and enhanced mineralization of FYM in the presence of urea.This rise may also be attributed to the transformation of ammonium into nitrate 66 .The decrease in the amount of available N from the tillering stage to maturity may be attributed to the absorption of N by plants and the loss of N through both living and non-living processes 67 .Furthermore, the ability of free-living N-fixing microorganisms is reduced as plants mature, primarily due to a decrease in the rate at which organic matter is deposited in the rhizosphere.Furthermore, the drop in nitrogen content at later stages could be attributed to a slowed rate of N mineralization, likely caused by a decrease in organic N and accumulation of hazardous metabolites.Mineralization can also be diminished due to the decline in labile organic matter and microbial activity in later phases 53 .Akmal et al. also documented the fluctuation in N content 68 , and Srinivasan et al. 66 .Zhang et al. 53 found that the simultaneous use of organic manure and fertilizer resulted in the maximum mineral N and NO 3 -N content levels during the tillering stage.These levels were lower at the transplanting stage and reached their lowest point during the grain-filling stage.The highest amount of N that may be obtained from organic manures is typically found at the late panicle initiation stage of the crop.This is because organic matter breaking down into minerals is slow during this time 69 .The tillering stage of the wheat crop has the highest need for N compared to the flowering and dough stages.An empirical correlation was found between the SOC and available N content of the soil, with a coefficient of determination (R 2 ) of 0.93 (Fig. 7).In addition, a polynomial correlation was detected between urease activity and the available N concentration of the soil (R 2 = 0.97; Fig. S2).

Available P
Applying FYM across both seasons resulted in a considerably increased amount (19.7 to 36.7%) of available P compared to applying it during a single season (Table 4).Applying organic manures, alone or in combination with N or NP fertilizers, resulted in a substantial increase in P availability in the soil 45 .Compared to the application of 10 t FYM ha −1 , applying 15 t FYM ha −1 significantly increased the availability of phosphorus by 9.7% and 12.1% at different stages of growth (Table 4).The box plot demonstrates that the lowest levels of accessible P were observed during stage 2. Additionally, 50% of available P values ranged from 39.4 to 74.2 kg ha −1 , as shown in Fig. 1F.The decrease in soil-available P during stage 2 (tillering) can be attributed to the increased need for the development of robust root systems, causing wheat plants to absorb a significant amount of P from the soil.Another potential factor could be the progressive provision of P resulting from the mineralization process of organic P derived from recently applied FYM.The decrease in P concentration following its peak could be attributed to using P by plants and the reduction in easily degradable organic phosphates 66 .The availability of P in soil is controlled by APA (alkaline phosphatase activity), and a polynomial relationship (R 2 = 0.96) was established between APA and the amount of available P (Fig. S3).Many other workers observed that applying organic manures led to increased available P content 2,54 .Applying 10 t ha −1 FYM in alfisols and alluvial soils resulted in a 13-16.9%increase in available P content compared to plots without FYM or fertilizer P application 12 .The use of FYM coupled with NPK fertilizers results in the release of organically bound P and solubilization of soil P through organic acids released during the breakdown of organic matter.The long-term application of FYM also resulted in a decrease in the activity of polyvalent cations such as calcium (Ca), iron (Fe), and aluminum (Al) due to chelation leading to a reduction in P fixation 70 .However, implementing INM ensures an increase in the availability of P due to the conversion of soil pH to a neutral level, improvement in soil structure, and positive effects of organic and inorganic fertilizer inputs on soil health 11 .In addition, the application of chemical N had a synergistic effect on P availability in soil, resulting in an increase of 7.5 to 9.4% in available P over the wheat-growing period (Table 4).The FYM 15 treatment outperformed all other treatment combinations in retaining available P in these soils for both seasons (Table 2).Conversely, Bahadur et al. 71 found that plots receiving fertilizer had the highest levels of available P. In contrast, there was no notable difference in available P among plots treated with various types of organic manure.Singh and Prakash 72 found that the highest accumulation of available P was achieved using INM.In another study, Akmal et al. 68 found that the levels of available P showed a notable increase from October to November, followed by a minor fall in December and reaching their highest value in January.An observed decline in available P was noted from January to April, followed by a minor change leading up to May.A strong correlation was found between the SOC and available P content of the soil, with a coefficient of determination (R 2 = 0.97) (Fig. 7).
Available S Sulfur primarily exists in organic form as ester sulfates, which are not available to plants and need to be hydrolyzed into inorganic sulfates before plants can absorb them.Arylsulfatase is an enzyme that catalyses the cleavage of the O-S bond and plays a significant part in the process of ester sulfate mineralization in soils.The applications of FYM, either alone or in conjunction with chemical fertilizers, impact aryl sulfatase activity.Applying FYM throughout both seasons considerably increased the amount of available S in soil ranging from 20.0 to 27.9% relative to the rabi season (Table 4).The application of 15 t FYM ha −1 was much better and increased the availability of S by 12.6 to 17.1% over the wheat growth season.The utilization of FYM resulted in an augmentation of microbial biomass and enzyme activities, leading to an overall improvement in the availability of S in soil 23 .
The plots treated with chemical N fertilizer exhibited a notable increase (8.2-9.7%) in the available S in the soil (Table 4).Like DOC, available N, and P, levels of available S were found to be highest at stage 3 followed by stage 4, stage 5, stage 1, and stage 2 (Fig. 1G).The lowest availability of S during stage 2 may be attributed to less mineralization caused by low temperature coupled with increased uptake by the crop.The subsequent rise in available S content may be attributed to enhanced mineralization from favorable temperatures and increased microbial activity in this semi-arid environment.Both seasons × FYM 15 significantly improved the availability of S compared to the other treatment combinations (Table 2).Regardless of the application season, S content was shown to increase with higher rates of FYM application.However, the effect of increasing N fertilizer was not significant.The heightened level of activity may be attributed to the conducive conditions for microbes that degrade carbon, as well as an improved ability to utilize substrates 73 .Basak et al. 20 also documented the superiority of integrated fertilization.The relationship between SOC and available S level in soil was polynomial (R 2 = 0.99) (Fig. 7).

Enzymes
The various stages of crop development require varying nutrient absorption, which results in fluctuating enzymatic activity in the soil 74 .

Dehydrogenase activity (DHA)
The soil enzyme dehydrogenase (DHA) is found in live cells, making it an indicator of the overall oxidative activity of soil microflora and a representation of microbial activity in soil 75 .When comparing the rabi season to the other seasons, applying FYM resulted in greater DHA levels.Specifically, in stage 1, stage 2, stage 3, stage 4, and stage 5, the DHA levels were 13.3, 21.9, 23.7, 21.0, and 10.4% higher, respectively (Table 5).Irrespective of the growth stages, the application of 15 t FYM ha −1 resulted in a considerable increase in DHA compared to 10 t FYM ha −1 treatment (Table 5).Using organic manures significantly increased DHA compared to mineral fertilizers and plots without any amendments 60 .The application of fertilizer N was crucial and considerably improved the DHA levels in wheat at various stages, increasing it from 11.0 to 23.2%.In rainfed wheat, adding NP resulted in a 22% increase in DHA compared to the control 64 .Overall, both seasons × FYM 15 exhibited a significantly higher capacity than the other treatment combinations in maintaining soil DHA levels (Table S1).Mandal et al. 16 previously demonstrated the highest DHA levels during the tillering stage of wheat.The maximum level of DHA in any crop is determined by the specific species of the crop being evaluated.In their study, Masto et al. 76 discovered that the highest levels of DHA were observed during the vegetative stage of maize cropping, and these levels were sustained until the flowering stage.However, DHA levels fell during later stages of growth, which aligned with biochar application to the maize crops.Our investigation found that the abundance of DHA followed a periodic pattern, with the highest levels observed at stage 3, followed by stage 4, stage 2, stage 5, and stage 1 (Fig. 1H).This pattern of abundance is similar to the availability of nutrients.Dash et al. 77 found that the administration of organic manure resulted in greater levels of DHA.These levels continued increasing up to 20 days of incubation but then dropped in aerobic and submerged environments.During a 60-year experiment, it was observed that the highest activity of soil DHA occurred during the heading stage of crop growth.However, after this stage, the DHA activity consistently declined.The highest DHA activity was recorded at different stages of crop growth when urea + PK + S + Zn-EDTA was applied, and the crops were covered with neem in the rice-wheat cropping system of the semi-arid region of Punjab 78 .After 47 years of long-term fertilization in humid subtropics of acidic alfisol in Ranchi under a soybean-wheat cropping system, the addition of NPK plus 0.40 t ha −1 lime resulted in 38.8% increase in DHA activity compared to the NPK treatment 79 .The peak activity observed at stage 3 can be due to the rise in the release of root exudates, which stimulates microbial activity.This activity gradually decreases as the crop reaches maturity 24 .The decline in maturity could be attributed to a gradual reduction in the decomposition rate caused by a lack of easily decomposable substrate towards the end of crop season 60 .The reduced DHA levels seen after harvest may be attributed to the soil's oxidation condition, which could be caused by limited moisture availability during maturation 68 .The combined use of organic manures and chemical fertilisers resulted in increased enzyme activity compared to using organic manures alone, as demonstrated by Bharti and Sharma 43 .An empirical correlation was found between the soil's SOC and DHA content, with an R 2 value of 0.97 (Fig. 7).
Vol:.( 1234567890 80 .Applying FYM in both seasons resulted in a considerable increase in β-Glucosidase activity in soil at all stages, compared to using it in a single season alone (Table 5).The application of 15 t FYM ha −1 had a considerably more significant effect on β-Glucosidase activity than the application of 10 t FYM ha −1 .The peak β-Glucosidase activity was at stage 4 of the wheat growth season.Furthermore, the general trend indicated a continual increase in activity up to stage 4 (Table 5).The box plot revealed that β-Glucosidase activity ranged from 262.5 to 441.0 µg PNP g −1 h −1 for 50% of the values at stage 4. In comparison, it ranged from 74.2 to 118.0 µg PNP g −1 h −1 at stage 1 (Fig. 1I).Fluctuations in enzyme activity can be caused by various variables, including changes in overall climate and local climate conditions, differences in nutrient availability, and alterations in the amount of leaf litter intake 77 .Piotrowska and Koper 81 discovered that the activity of the β-glucosidase enzyme dramatically increased as the rate of FYM rose.However, they did not find any significant impact of N fertilization on enzyme activity, particularly at higher dosages of FYM in winter wheat crops.Adding 200 kg N per hectare boosted the levels of β-glucosidase in a long-term N fertilization experiment conducted on a wheat-soybean system 18 .The increased activity of enzymes that break down cellulose and hemicellulose is caused by the favourable conditions for microorganisms that degrade carbon, which is created by adding FYM 73 .A strong correlation (R 2 = 0.99) was found between SOC and β-Glucosidase activity, indicating a polynomial relationship (Fig. 7).The combination of FYM 15 × N 120 had a considerably more significant effect than the other combinations at stage 2 and stage 5 (Fig. 3).Sharma et al. 82 also found that the enzyme activity was maximum when FYM was used in conjunction with fertilizers, compared to using fertilizers alone.

Urease activity
Urease activity in soil serves as a biological indicator and is primarily emitted by plants, with a smaller contribution from soil microbes and animals 83 .Urease facilitates the more effective handling of urea fertilizers by catalyzing the hydrolysis of urea-containing fertilizers into CO 2 and NH 3 .The soil's physical and chemical characteristics and agricultural practices affect the enzymatic activity 84 .The 15 t FYM ha −1 treatment resulted in a significant increase in urease activity ranging from 10.1 to 17.0% at different stages (Table 5).Introducing organic manures led to a considerable rise in urease activity 85 .The higher enzyme activity seen with the increased application rate of FYM can be related to the augmented populations of microorganisms resulting from greater carbon availability, leading to the release of additional extracellular enzymes 13 .The highest level of urease activity (49.8 µg N g −1 h −1 ) was observed while using a combination of 10 t FYM and 5 t compost per hectare, compared to using mineral fertilizer alone 86 .The trend of urease activity over the growing phase of wheat followed the order stage 3 > stage 4 > stage 5 > stage > stage 2 (Fig. 1J).The first decrease in enzyme activity may be attributed to the low temperature and the negative correlation between urease activity and temperature, as previously documented by Akmal et al. 68 .The increased urease activity observed in later stages can be due to higher N content, greater availability of organic matter, faster breakdown, and release of ammonium 45 .Both seasons × FYM 15 had a considerably higher capacity for enhancing urease activity compared to the other combinations (Fig. 4).Several researchers have found that the activity of urease in soil reduces over time when urea/N fertilizers are used, relative to unfertilized soil.This is because soil microbes absorb mineral N from fertilizers 87 .In the rain-fed soybean-wheat system, the urease activity was suppressed in soils that were amended with organic material after 33 years of fertilization.The highest urease activity was recorded in unamended plots followed by plots that received NP treatments 88 .Akca and Namli 89 found that increasing the dose of poultry litter biochar to 600 kg ha −1 decreased enzyme activity.Patel et al. 13 have demonstrated that combining organic manures with mineral fertilizers leads to increased urease activity compared to using organic manures alone.Our study showed a polynomial correlation (R 2 = 0.95) between SOC and urease activity in semi-arid soils (Fig. 7).Additionally, a correlation between urease activity and available N was also identified with an R 2 value of 0.97 (Fig. S1).

Alkaline phosphatase activity (APA)
Phosphatase refers to a class of enzymes that catalyse the hydrolysis of esters and anhydrides of phosphoric acid and have a significant impact on the P cycle by liberating P. Phosphatase enzymes encompass both active and stable extracellular enzyme activity, thereby representing the maximum level of phosphatase activity in soils 90 .The higher level of APA seen in soil treated with manure and straw residues can be attributed to increased microbial activity and potentially a wider variety of phosphate-solubilizing bacteria resulting from the long-term addition of manure 91 .A notable rise in APA (10.6, 5.2, 7.2, 9.3, and 8.9% at wheat growing stages 1 to 5, respectively) was observed when treated with FYM 15 .Chemical N treatment, similar to the available P, considerably enhanced the abundance of active phosphatase enzymes in the soil (Fig. 5).Hota et al. 92 reported more excellent alkaline phosphatase activity in NP treatment compared to the absolute control.The decrease in soil pH caused by organic acids generated during the decomposition of organic matter leads to an increase in enzyme activity.The FYM serves as a supply of C, N, and P for soil microbes, potentially enhancing the activity of APA.At stage 5, the APA was elevated by 15.4 to 24.7%, 19.4 to 22.0%, and 20.5 to 21.4% under the modes of FYM and chemical N levels, respectively, compared to stage 1 (Fig. 5).Prior studies 85 have indicated that the application of organic manures leads to higher APA compared to chemical fertilizer and control.Additionally, APA increased with higher dosages of fertilizer.The present study found that both seasons and the degree of FYM had a significant and positive impact on APA (Fig. 6).The enzyme activity decreased with a higher dose of chicken litter biochar.However, the maximum enzyme activity was observed in the INM system compared to using organic materials alone.This finding is supported by studies conducted by Moharana et al. 64 and Akca and Namli 89 .Our study found a strong correlation between SOC and APA with an R 2 value of 0.99 (Fig. 7).Additionally, a high correlation was identified www.nature.com/scientificreports/ between APA and P with an R 2 value of 0.96 (Fig. S2).Throughout the wheat's growth phase, the largest APA was seen during stage 3, followed by stage 2, stage 4, stage 5, and stage 1 (Fig. 1 K).The shift of soil pH towards alkaline levels increased APA due to soil alkaline conditions combined with N management measures.Seasonal fluctuations, plant developmental stage, and organic matter content are significant factors that strongly impact soil microbial biomass.DeForest et al. 93 determined that the activity of enzymes is influenced by the initial bioavailability of P in soil.Islam and Borthakur 24 have observed increased phosphatase activity during the robust growth stage, followed by a drop after maturity.The soil parameters exhibited their highest values at different stages of the wheat growing period.Specifically, the highest values of soil pH, EC, SOC, DOC, available N, P, S, DHA, β-Glucosidase activity, urease, and APA were seen at stage-1, stage-2, stage-2, stage-3, stage-3, stage-3, stage-3, stage-3, stage-4, stage-3, and stage-3, respectively.Prior research has demonstrated that the activity of enzymes in wheat increased from the time of sowing to the flowering and intense growth stages, and subsequently decreased at the time of harvest 21,23

Principal component analysis (PCA)
The principal component analysis (PCA) effectively distinguished the soil variables and treatments in a perpendicular space (Fig. 8).PC1 and PC2 accounted for 97.8% of the variability in the data set, with PC1 explaining 94.7% and PC2 explaining 3.15% of the overall variability.The first principal component (PC1) had an eigenvalue of 10.41, whereas the second principal component (PC2) had an eigenvalue of 0.35.Each soil variable was associated with a factor loading or eigenvector weight value, indicating its contribution to variability (Table S2).In PC1, the DHA had the highest loading value of 0.307, closely followed by available sulphur and urease activity, with a loading value of 0.306.Additionally, the DHA showed significant correlations with other markers with high loading values.In the second PC (PC2), the indicator with the highest loading value was EC (− 0.682), followed by SOC (0.406) and APA (0.308).The proportion of total variance computed based on the weight of each principal component (PC) indicates that PC1 accounts for 97% of the variance, while PC2 accounts for 3%.The PCA biplot clearly showed that applying 15 t FYM ha −1 along with 120 N ha −1 during both seasons was distinctly separated and identified as the most effective treatment compared to the other combinations in this long-term study conducted in a semi-arid location (Fig. 8).

Conclusion
The long-term seasonal application of farmyard manure (FYM) and fertilizer nitrogen (N) substantially impacted the soil parameters.During the 51st cycle of wheat under pearl millet-wheat cropping, the impact of FYM application in different seasons on soil properties in sandy loam soils exhibited the following order: both seasons (combined treatment in both rabi and kharif seasons) had the most significant impact, followed by rabi season application, and then kharif season application.Although all three factors contributed to an increase in soil electrical conductivity (EC), the levels remained significantly below the permissible threshold of 0.80 dS m −1 for crop production in these sandy loam soils.The application of 15 t of FYM ha −1 exhibited considerably more significant levels of dissolved organic carbon (DOC), available P and S, than that of 10 t FYM ha −1 .The application of 15 t FYM significantly increased the availability of N compared to the application of 5 t FYM ha −1 .Irrespective of the stages, every level of FYM exhibited positive and discernable impacts on soil properties.The application of FYM 15 Fig. 8.The plot of principal component analysis (PCA) on the soil properties, including soil pH, electrical conductivity (EC), soil organic carbon (SOC), dissolved OC (DOC), available N, P, S, dehydrogenase activity (DHA), β-Glucosidase (Beta glu), urease and alkaline phosphatase activity (APA) in soils (pooled data of five growth stages of wheat) under pearl millet-wheat cropping system in North-western, India.PC = principal component.
enhanced DHA levels and urease activity considerably and was more effective for strengthening β-Glucosidase activity and APA activity than the FYM 10 treatment.The application of fertilizer N significantly contributed to the accumulation of SOC and enhanced the replenishment of available nutrients (N, P, and S) in the soil solution.At various phases of wheat growth, the application of 120 kg N significantly improved DHA, β-Glucosidase, urease, and APA activity compared to no application of N. The interaction between the application of FYM 15 in both seasons and N 120 significantly increased the content of DOC and β-Glucosidase activity compared to other combinations.The availability of N was highly and positively correlated with urease and APA activity.The PCA biplot clearly showed that applying 15 t FYM ha −1 and 120 kg N ha −1 over both seasons was distinctly separated and was considered the most effective compared to the other treatment combinations.In PC1, the DHA had the highest loading value of 0.307, closely followed by available sulphur and urease activity, with a loading value of 0.306.Applying 15 t FYM and 120 kg N ha −1 proved the most effective options for improving soil quality and crop growth in sandy loam soils.It resulted in the highest organic carbon (OC) levels, increased nutrient availability, and enhanced activity of beneficial enzymes throughout the growth stages of wheat in a pearl millet-wheat cropping system. https://doi.org/10.1038/s41598-024-72076-w

Fig. 3 .Fig. 4 .
Fig. 3. Effect of level of FYM × fertilizer N on β-Glucosidase activity at different growth stages of wheat.

Fig. 5 .Fig. 6 .
Fig. 5.Long-term impact of mode, level of FYM and fertilizer N application on alkaline phosphatase activity (APA) at different growth stages of wheat.

Table 2 .
Impact of mode × level of FYM on soil organic carbon (SOC), available P and S at different growth stages of wheat.The subscript figures in treatment indicate the dose of FYM in t ha −1 .

Table 3 .
Long-term impact of mode, level of FYM, and fertilizer N application on dissolved organic carbon (DOC: mg kg −1

Table 4 .
Long-term impact of mode, level of FYM, and fertilizer N application on available nutrients (NPS: kg ha −1 ) at different growth stages of wheat.Note: The subscript figures in treatment indicate the dose of FYM in t ha −1 and the dose of fertilizer nitrogen in kg ha −1 .

Table 5 .
Long-term impact of mode, level of FYM, and fertilizer N application on soil enzyme activity at different growth stages of wheat.The subscript figures in treatment indicate the dose of FYM in t ha −1 and the dose of fertilizer nitrogen in kg ha −1 .